STRUCTURAL DYNAMICS OF TELOMERIC OVERHANG ACCESSIBILITY
BY
HELEN HWANG
DISSERTATION
Submitted in partial fulfillment of the requirements
for the degree of Doctor of Philosophy in Bioengineering
in the Graduate College of the
University of Illinois at Urbana-Champaign, 2013
Urbana, Illinois
Doctoral Committee:
Assistant Professor Sua Myong, Chair
Professor Brian Cunningham
Professor Brian Freeman
Assistant Professor Jian Ma
Abstract
Telomeres are dynamic nucleoprotein complexes that cap and protect chromosome
ends from deleterious degradation and fusion events. In most eukaryotes, telomere
length is regulated by a basal level of telomerase, a specialized reverse
transcriptase, which can add telomeric repeats to the 3’ end of chromosomes. In
normal cells, the telomere is shortened due to the end replication problem and DNA
degradation, which sets the cell lifespan. 85% of cancer cells can escape this growth
limitation by upregulating telomerase. The remaining cancer cells activate an
alternative lengthening of telomere pathway (ALT). Therefore, treatments that
target the telomere itself would potentially disrupt both mechanisms that cancer
cells use to sustain unlimited proliferation. Hence, the telomere is an attractive
target for anti-cancer drugs.
Human telomeres terminate with an overhang repeat sequence that can self-fold
into a G-quadruplex structure, allowing regulation of the telomere overhang. In
addition, telomeric-binding proteins such as POT1 and TPP1 can sequester the
overhang to regulate telomerase activity in vivo. Furthermore, existing small
molecules that bind G-quadruplex have been shown to inhibit telomerase activity.
However, there is little evidence on how the telomere-binding proteins impact the
overhang structure.
Previous structural studies have only provided snapshots of static telomere states,
but have not provided any dynamics that may be exhibited. Bulk solution studies are
unable to differentiate between the folded and the unfolded G-quadruplex states, as
well as transient protein-bound states and intermediates.
ii
With a single molecule FRET system, we can distinguish between folded, unfolded,
and intermediate states of individual telomeric overhangs. Here, I present the study
of structural dynamics of individual telomeric overhangs by itself and with
associated proteins in real time by single molecule fluorescence detection.
Accessibility of the telomere depends highly on the number of telomeric repeat. In
addition, telomere-associated proteins like POT1 can bind the telomeric overhang
independent of repeat number, while POT1-TPP1 can actively slide on the 3’ end of
the overhang. Furthermore, we have developed a fluorescence telomerase assay
that can measure the binding, extension, and dissociation events of a single
telomerase. Using this technique, we observe that the dynamic flexibility of the
telomeric overhang facilitates telomerase access and leads to telomere lengthening.
Ultimately, we envision that the understanding of the structure and dynamics of the
telomere with its partner proteins will enable us to design and screen drugs that
specifically target the telomeric overhang to regulate the telomere lengthening and
immortal phenotype of cancer.
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To my friends and family
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Acknowledgements
I would like to thank my advisor, Sua Myong, for her support and continual guidance
on my education, career, and life throughout my graduate study. She has taught me
to think like a scientist and has nurtured a positive creative lab environment. Her
creativity, profound insight, and enthusiasm for teaching, scientific research, and on
life have been truly inspirational.
I would also like to thank Professors Brian Cunningham, Brian Freeman, and Jian Ma
for serving on my committee and offering constructive criticisms. Also, the
Department of Bioengineering including Wendy Evans, Samuel Smucker, and
Professor Michael Insana have made my time at University of Illinois enjoyable.
They have been a pleasure to work with.
Peggy Qiu, Alex Kreig, Ramreddy Tipanna, Younghoon Kim, Hye Ran Koh, Cong Xu,
Jacob Calvert, and Shirley Wang have been wonderful comrades who have provided
invaluable insights and technical assistance. Their constant energy and passion
continually energized our lab environment and have always uplifted my spirits.
I would also like to thank Taekjip Ha and the Ha lab members that have offered
innumerable technical advice, especially Ankur Jain, Jeehae Park, Hajin Kim, Prakrit
Jena, Xinghua Shi, Sultan Doganay, Yuji Ishitsuka, Ben Leslie, and Jingyi Fei.
Patricia Opresko, Noah Buncher, and Justin Lormand have been excellent
collaborators and have been a pleasure to work with. I am extremely grateful for
their generosity of precious substrates and proteins and also their scientific insights.
Finally, I would like to thank my family and friends for their continual support
through my graduate career and Johan Rhodin for putting up with all my craziness.
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Table of Contents:
I: INTRODUCTION....................................................................................................................1
1.1
A short history of telomere biology......................................................................1
1.2
Telomeric DNA sequences…....................................................................................2
1.3
The Hayflick limit and the end replication problem.......................................3
1.4
Telomeric binding proteins.....................................................................................4
1.4.1 A specialized reverse transcriptase enzyme, telomerase............................4
1.4.2 ALT pathway proteins..............................................................................................5
1.4.3 Shelterin Complex.....................................................................................................6
II: SINGLE MOLECULE FLUORESCENCE TECHNIQUES...................................................8
2.1 FRET as a ratiometric distance measure...................................................................8
2.2 Single fluorescence as a short distance measure...................................................9
2.3 Single Molecule Pulldown to Study Complex Protein Complexes..................10
III: TELOMERE OVERHANG ACCESSIBILITY DEPENDS ON REPEAT NUMBER....12
3.1 Introduction.....................................................................................................................12
3.2 Telomere overhangs longer than four repeats display multiple
conformations and dynamics............................................................................................15
3.3 GQ folding limits overhang accessibility to complimentary DNA..................18
3.4 RAD51 and RPA binding depends on repeat number........................................21
3.5 WRN and BLM display repetitive unfolding of GQ...............................................24
3.6 POT1 binding is independent of repeat number.................................................27
3.7 Telomerase binding and extension activity depends on repeat number…28
3.8 Discussion.........................................................................................................................30
3.9 Methods and Materials.................................................................................................32
IV: POT1-TPP1 REGULATES TELOMERIC OVERHANG STRUCTURAL
DYNAMICS................................................................................................................................41
4.1 Introduction.....................................................................................................................42
4.2 POT1 binds the telomeric overhang sequentially one OB fold at a time.....44
4.3 POT1 binds one OB fold at a time..............................................................................48
4.4 POT1-TPP1N induces dynamic folding and unfolding of telomeric
overhangs.................................................................................................................................50
4.5 POT1-TPP1N displays dynamic movement on telomeric overhangs...........53
4.6 POT1-TPP1N Slides on Mutant Telomeric Sequence..........................................56
4.7 Discussion.........................................................................................................................60
4.8 Methods and Materials.................................................................................................63
V. A REAL-TIME SINGLE TELOMERASE EXTENSION ASSAY .....................................68
5.1 Introduction.....................................................................................................................68
5.2 Development of a Real Time Single Telomerase Extension Assay.................71
5.3 Telomerase assembly is dependent on dNTP concentration..........................72
5.4 POT1-TPP1 promotes telomerase extension and dissociation......................73
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5.5 Sliding of telomeric overhang POT1-TPP1 in the presence of
telomerase...............................................................................................................................74
5.6 Discussion.........................................................................................................................75
5.7 Methods and Materials.................................................................................................76
VI: CONCLUSIONS AND PERSPECTIVE.............................................................................80
6.1 Accessibility of telomeric overhang by ALT proteins and telomerase.........80
6.2 Why does POT1-TPP1 slide on DNA?.......................................................................80
6.3 Telomerase activity can be measured as a function of binding and
extension..................................................................................................................................81
6.4 Long term goals...............................................................................................................82
REFERENCES............................................................................................................................84
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I.
INTRODUCTION
1.1
A short history of telomere biology
In the 1920s, Hermann Muller (Nobel laureate 1946) studied chromosome breaks
like inversions, translocation, and deletions, by radiating fruit flies with x-rays (1).
Muller observed that he could recover that the chromosome breaks with his genetic
methods. However, he was unable to recover the chromosome terminal deficiencies.
He reasoned that the recovered chromosomes were usually the result of the
rejoining of two broken ends and such rejoining could not occur between originally
free ends or between broken ends and originally free ends. Thus, in 1938, Muller
and his colleague Darlington, coined the term “telomere” for the free end of the
chromosome.
At the same time, Barbara McClintock (Nobel laureate 1983) studied genetics in
maize. She developed techniques to visualize individual maize chromosomes, and
observed that there are knobs located at the chromosome ends. She recognized that
these knobs may be important and named them nature ends’ of the chromosomes
(2) . In agreement with Muller, McClintock pointed out that broken ends could not
fuse with natural ends. In addition, McClintock noticed that broken ends could be
“permanently healed” in some early embryo tissues. Based on her observations, she
reasoned there must be a mechanism to heal a single broken end “during the
replication cycle of the chromosome (3) . McClintock’s insight pioneered the
1
modern understanding that telomerase, an enzyme, is actively involved in healing
broken ends during S-phase.
1.2
Telomeric DNA sequences
Telomeric DNA in humans is mostly double stranded DNA (dsDNA) repeat
sequences (2000 repeats) that terminate with a 3’ single stranded (ss) DNA
overhang. The overhang provides a primer substrate for extension by telomerase
and for a binding site to the specific telomeric proteins. In humans, the overhang is
estimated to be 100-200 nucleotides long of tandem TTAGGG repeats ((4) (4) (5)).
In the 1980s, biochemical studies showed that runs of adjacent guanines as seen in
telomeric DNA can spontaneously fold into a complex secondary structure, a Gquadruplex (6). These G-quadruplexes are guanine quartets that arise from
hydrogen bonding called Hoogsteen base pairing, where each guanine base makes
two hydrogen bonds with its neighbor. This interaction is strongly coordinated by
monovalent cations like K+ and Na+ and hence physiological conditions favor their
formation (Figure 1a, 1b). There is increasing evidence that G-quadruplexes exist in
vivo (7), (8) and limit telomerase extension in vitro (9, 10). However, it is a
challenge to interrogate the G-quadruplex in vivo since it is difficult to knock-out a
DNA structure using genetics to determine the resulting phenotype. While a Gquadruplex can be exchanged with a novel sequence, the novel repeats do not bind
telomeric proteins, and so the phenotypes are less representative (11).
2
Figure 1 A. G-quadruplex structure is mediated by monovalent cations and Hogsteen base pairing. B.
The guanines stack into a planar tetrad structure.
1.3
The Hayflick limit and the end replication problem
Historically, it was believed that vertebrate cells had an unlimited potential to
replicate. This was because scientists may have only looked at cells that contained
embryological cells or cancerous cells like HeLa (a human cervical carcinoma in
1952) and L cells (from mouse mesenchyme in 1943), in which the cells seemed to
be able to replicate indefinitely. In 1965, Leonard Hayflick with Paul Moorehead
demonstrated that normal human fetal cells could divide only 40 to 60 times, before
entering a senescence phase, a phase where cell growth diminishes and cell division
stops.
The end replication problem exists due to the properties of DNA polymerase of 1)
unidirectional growth and 2) the requirement for a primer to initiate synthesis.
However, since DNA is linear and requires a RNA primer, each daughter strand
would be shortened on the 5’ end of the new DNA strand after replication (12) .
3
Unicellular organisms and viruses have evolved special mechanisms to overcome
the end-replication problem. Due to the circular nature of their genome, they can
simply eliminate the ends and the primers.
However, in the absence of a telomere maintenance system, eukaryotes lose about
terminal sequences of 3-5 base pairs/end/division, which is a rate predicted by the
end replication problem (13). Furthermore, mouse and human telomeres shorten
much faster with an estimated predicted rate of 50 to 150 basepairs/end/cell
division which is likely due to UV damage and exonucleases (14) .
1.4
Telomere binding proteins
1.4.1 A specialized reverse transcriptase enzyme, telomerase
Figure 2 Telomerase extension involves three steps – binding, elongation, and translocation
To solve the end replication problem, eukaryotic systems have evolved with the use
of an enzyme named telomerase, which can maintain telomere length and
counteract incomplete replication (15-17). In 85% of cancer cells, loss of telomeric
4
DNA due to degradation and incomplete replication is balanced by telomere
enlongation via telomerase. The telomerase enzyme is a ribonucleoprotein that
contains two essential core components, a telomerase reverse transcriptase (TERT)
and a telomerase RNA (TR). The RNA region contains a complementary region to
the telomeric repeat sequence, allowing conventional Watson-Crick base pairing to
specify the telomeric sequence. The telomerase reaction works by adding
deoxynucletide (dNTP) triphosphates onto the 3’ end of telomeric DNA sequences.
It is thought the telomerase then translocates and realign and the new 3’-end of the
telomere template, and the process is repeated (Figure 2).
1.4.2 ALT pathway proteins
Although the majority (85%) of cancers activate telomerase to maintain their
telomere lengths, some cancer cells (15%) and tumors are able to maintain their
telomere lengths over many populations in the absence of telomerase activity
through a mechanism termed alternative lengthening of telomeres (ALT) (18) . To
date, ALT has only been detected in cancer cells and immortalized cell lines.
However, there is indirect evidence that normal splencocytes have ALT activity and
elongated telomeres in vivo in telomerase-null mice (19). Aside from being able to
be immortalized without telomerase activity, ALT cells are characterized by their
heterogeneous and dynamic telomere length distribution phenotype. Fluorescence
in situ hybridization (FISH) studies with a telomere-repeat-specific probe show that
there is a heterogeneous telomere length phenotype within one cell (20) (21) and
the telomere length fluctuates significantly during cell proliferation (22). They
5
concluded that the rate of telomere length changes were proportional to the
frequency of chromosome fusion events, which is consistent with a homologous
recombination mechanism, however the exact mechanism still remains unknown.
1.4.3 Shelterin complex
Figure 3 The Shelterin complex
At the telomeres, there is a complex of at least six-telomeric specific proteins that
are only localized to the telomere, called the Shelterin complex (Figure 3). This
complex is made up of at least two proteins that bind to ds telomeric DNA (TRF1,
TRF2) and a third, POT1, that binds to TTAGGG repeats in single stranded form.
Together with other protein-interactions, give the telomeric complex its specificity
for the sequence and structure of telomeric DNA. Stoichiometric studies suggest that
the shelterin complex is abundant on chromosome ends and increases as telomere
lengthen (23) (24). The Shelterin proteins exist only at the telomeres throughout
the cell cycle and their function is limited to the telomeres. When the Shelterin is
removed, the telomeres are susceptible to attack by DNA damage by six different
pathways ranging from ataxia telangiectasia (ATM) and ATR, and repair mediated
through non-homologous end-joining (NHEJ) and homology-directed repair (HDR)
pathways (25) (26). Hence, the Shelterin complex protects chromosome ends from
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DNA damage surveillance and repair pathways and are important in maintaining
telomere length homeostasis.
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II.
SINGLE MOLECULE FLUORESCENCE TECHNIQUES
Single molecule spectroscopy can be used to study dynamic structural properties of
DNA with secondary structures, DNA-protein interactions, and stoichiometry. Due
to the dynamic properties of telomeric DNA into a folded and unfolded Gquadruplex, FRET is a convenient method to study the interconversion between
states as well as the different intermediate states that may occur when proteins
bind.
2.1
FRET as a ratiometric distance measure
Figure 4 Two FRET schema
There are many great reviews on single molecule Fönster Resonance Energy
Transfer already (27) (28). Here, I will discuss two common FRET schema – one
where the two cyanine dyes are located on DNA and one where the cyanine dye is
located on the protein and the other on DNA. The most simple FRET scheme is
when the DNA is labeled in two distinct locations with the fluorescent dyes. This is
ideal for real time monitoring changes in the persistent length or folding into
8
secondary structure, for example the folding-unfolding of the G-quadruplex in the
telomeric overhang (Figure 4A) (29) (30) (31).
Another scheme with FRET is where the protein is labeled with a dye and the DNA
is labeled (Figure 4B). In this manner, the distance between the dye on the DNA
and the protein can be monitored. If the protein binds or exhibits dynamic
behavior, it will be reflected in the single molecule traces as well as the overall
FRET histograms. In a protein slides on DNA, the readout will be fluctuating FRET
traces.
2.2
Single fluorescence as a short distance measure
Figure 5 A PIFE experiment – the DNA is fluorescently labelled while the protein is label-free.
Although FRET is a useful method to study transient interactions and dynamic
behaviors, fluorescent tagging of proteins can be inefficient, difficult, and
sometimes perturbs protein function. Furthermore, study of proteins with high
dissociation constant, Kd requires addition of high concentration of fluorescence,
which can hinder the detection of single molecules. The Protein Induced
Fluorescence Enhancement (PIFE) assay is a single fluophore assay which bypasses
the labeling of proteins since the fluorophore attached to substrate serves as a
reporter of the protein binding and its movement (Figure 5). The intensity of a
9
fluorophore is enhanced upon binding of a protein it its vicinity. With the same
microscopy setup used to monitor FRET at the single molecule level, PIFE can be
employed to study protein-nucleic acid interactions simply by measuring the
fluorescence intensity (32) (33) (34). As part of my master’s work, we have
characterized the distance sensitivity of PIFE and demonstrated that it can
complement FRET by providing information in the short distance-sensitivity range,
where FRET is insensitive (Figure 6) (35).
Figure 6 Distance sensitivity of PIFE and FRET
2.3 Single Molecule Pulldown to Study Complex Protein Complexes
The single molecule pulldown assay (SiMPull) is a newly developed easy-to-use
method for probing single macromolecular complexes directly from cell or tissue
extracts (36, 37). This method immobilizes a macromolecule using biotinaylated
antibodies onto a passivated surface when cell lysate is applied. The unbound
components can then be washed away, and single molecule florescence microscopy
can be applied. Primary and secondary antibodies can be used to confirm the
10
presence and isolation of a protein of interest. Due to the complex nature of
telomerase, SiMPull is ideal for the study of telomerase.
11
III.
TELOMERE OVERHANG ACCESSIBILITY DEPENDS ON REPEAT NUMBER
The work in this chapter is under review for publication. It examines how the
telomeric DNA overhang can directly regulate binding of telomere-binding proteins
and telomerase. The G-rich single stranded DNA at the 3’ end of human telomeres
can self-fold into G-quaduplex (GQ). However, telomere lengthening by telomerase
or recombination-based alternative lengthening of telomere (ALT) mechanism
requires protein loading on the overhang. We report here that telomeric overhangs
exhibit dynamic properties when the length exceeds four TTAGGG repeats.
Overhangs with four and eight repeats that can fold into one GQ and two GQs,
respectively, show limited accessibility to telomerase loading and extension activity,
and to loading of the ALT-associated proteins RAD51, RPA, WRN and BLM.
However, overhangs with five to seven repeats are much more accessible to these
proteins. In contrast, POT1, the telomere-specific single-stranded DNA binding
protein, binds independently of repeat number. Our results suggest that the
telomeric overhang repeat length and dynamics may contribute to telomere
extension via telomerase action or the ALT mechanism.
3.1 Introduction
Human telomeres terminate in a 50-200 nucleotide single-stranded 3’ overhang that
plays a pivotal role in chromosome end protection (4) (4) (5). The G-rich overhang
serves as the substrate for telomerase which extends telomeres by adding tandem
TTAGGG repeats and as a binding site for the POT1 protein which prevents the
12
activation of the checkpoint kinase ATR, inhibits sister chromatid fusion, and
represses homologous recombination (38) (39) (40) (41).Furthermore, the
telomeric overhang invades the duplex region forming a t-loop (42) and thereby
blocking ATR and ATM signaling which is essential to prevent the chromosome end
from being incorrectly recognized as a DNA double strand break(43).
Owing to the end-replication problem, the ends of chromosome shorten with each
round of DNA replication in all somatic cells (44) (45). The progressive telomere
attrition results in replicative senescence, growth arrest and apoptosis (46) (47).
Cancer cells, however overcome this barrier mostly (85-90%) by upregulating
telomerase activity to sustain the telomere length (48, 49). Nevertheless, a
significant subset (10-15%) of cancer cells are telomerase-negative, and lengthen
telomeres by a telomerase-independent mechanism, termed Alternative
Lengthening of Telomeres (ALT) (50) (51) (52). ALT is likely mediated by
homologous recombination (HR) mechanisms (53) (54) (55). Therefore, all cancer
cells exhibit indefinite lifespan supported by either the telomerase-catalyzed
extension or ALT-mediated lengthening of telomeres. Due to this common feature of
all cancers, telomeres and telomere-associated proteins have been attractive drug
targets for anti-cancer therapy (49) (56) (57).
The telomeric overhang presents a highly vulnerable target which can be
misrecognized to induce a DNA damage response that can lead to chromatin fusion
and erosion (58) (59) (44) (60) (61) . Therefore, the capping and protection of the
13
telomeric overhang is of utmost importance for genome integrity. From
Schizosaccharomyces pombe to humans, the overhang is specifically bound by the
shelterin component POT1, which prevents ssDNA stimulated repair and
recombination (38) (62) (Denchi and de Lange, 2007; Hockemeyer et al., 2005).
Furthermore, POT1 regulates access to the overhang to facilitate complete
chromosome replication (63) (64) (65, 66).
Recent reports suggest a complex relationship between DNA repair proteins and
human telomeres(58). For example, numerous proteins involved in telomere
function, HR, DNA damage repair and DNA replication are associated with ALT (67).
While many of these proteins need to gain access to the telomere overhang,
successive repeats of TTAGGG can fold into G-quaduplex structure, which may serve
as a structural barrier to protein access. Using highly sensitive single molecule
techniques, we have examined the folding behavior of overhangs with varying
numbers of telomeric repeats (4-8) and how this behavior relates to overhang
accessibility to proteins involved in telomere processing. We chose to investigate
both the ALT pathway associated molecules including a C-circle mimic of
complementary DNA (C2), RAD51, RPA, Werner (WRN), Bloom (BLM) and also
POT1 and telomerase, both of which bind telomere overhangs in a sequence specific
manner. Our results reveal that sequences with four (G4) and eight (G8) repeats of
TTAGGG are likely to fold into one GQ and two GQs, respectively, thus limiting
protein access. In contrast, five to seven repeats exhibit significantly higher
accessibility to DNA and most protein molecules tested. Importantly, the same
pattern of accessibility was shown in telomerase extension activity. Contrary to
14
ALT-associated proteins and telomerase, POT1 binds to all telomere overhang
lengths with no bias, suggesting an active binding mechanism that does not rely on
the overhang accessibility. Our observations point to the importance of the
dynamics of G-quadruplex structures located at 3’ overhangs in governing the
processing of telomeres by proteins involved in telomere lengthening mechanisms.
3.2 Telomere overhangs longer than four repeats display multiple
conformations and dynamics
Figure 7 (A) Schematic of DNA used for single molecule FRET. Two dyes, Cy3 and Cy5
are attached to both sides of 4 repeats of TTAGGG. High FRET and low FRET
correspond to folding and unfolding of G-quadruplex (GQ), respectively. (B) Single
molecule frequency histograms of DNA with 4, 5, 6, 7, and 8 repeats of TTAGGG. The
red outline is the summation of 1, 2, and 4 Gaussian fits for G4, G5, and G6
respectively. (C) Representative single molecule trace for G4-G8. (D) Plot of half time,
t1/2 obtained from fitting dwell times to exponential decay. (dwell times are denoted
by blue arrow in single molecule trace)
Although numerous studies have examined the folding kinetics of human Gquadruplex (GQ), only a few used physiologically relevant telomeric substrates that
15
have more than four repeats of TTAGGG (68). Melting temperature and circular
dichroism experiments have suggested that GQs are blocks that resemble “beadson-a string” and that they can move independently of each other and being
constrained only by connecting linkers (69) (70). In addition, telomeric repeats in
multiples of four result in increasing enthalpy and entropy arising from stable GQ
formation (70). On the contrary, a previous AFM study showed that long telomeric
3’ overhangs did not form the maximum number of GQs. Rather, they were
dispersed suggesting that GQ folding may be dynamic (71).
To investigate the effect of telomere repeat length on GQ conformation, we prepared
a series of 3’ overhang DNA substrates with varying numbers of TTAGGG repeats
from 4 to 8, which we refer to as G4-G8 hereafter. Each DNA was labeled with two
fluorescent dyes, Cy3 and Cy5 at both ends of the ssDNA for measuring FRET
(Fluorescence Resonance Energy Transfer). FRET, as a molecular ruler, reports on
the folding status of the telomeric overhang such that a high FRET signal indicates
GQ folding in G4 DNA (Fig. 7a). In 100 mM KCl, we obtained one, two, and four
FRET peaks for four (G4), five (G5), and six (G6) telomeric repeats respectively (Fig.
7b). Each FRET histogram was built from FRET values of over 5000 individual
molecules. We interpret the discrete FRET peaks as reflecting formation of one, two
and four conformations in G4, G5 and G6, respectively. With seven telomeric repeats
(G7), we observed a broad FRET histogram with no apparent peaks, likely due to
formation of multiple conformations. For G8, we also obtained a broad FRET
distribution, yet with a prominent high FRET peak. Based on the total length of
16
single stranded DNA of 48 nucleotide in G8, this high FRET cannot be due to the ion
induced compaction(72), but likely results from the formation of two GQs from two
sets of four repeats (Fig. 7b). Representative single molecule traces show that G4
stays in one high FRET state while the G5 and G6 overhang displays two and four
FRET states, respectively, likely representing different conformations arising from
alternative folding of GQ. G7 and G8 traces exhibit many more FRET states in
exchange, reflecting dynamic exchanges amongst multiple conformations (Fig. 7c).
Consistent with the FRET histograms, the DNA constructs with increased repeat
numbers involve more FRET states. In addition, the rate at which conformers
exchange is higher for longer overhangs. To quantify this effect, we collected dwell
times from about 1000 molecules for each DNA and fitted the data to an exponential
decay. The halftime, T1/2 obtained from the fit reveals shorter times for longer
overhangs suggesting that increased repeat number induces faster dynamics (Fig.
7d). Taken together, telomeric overhangs longer than four repeats exhibit dynamic
folding states. Longer lengths induce more complex folded conformations and faster
dynamics. Interestingly, despite the fast conformational dynamics, overhangs with
eight repeats display frequent transitions to the high FRET state, likely arising from
the formation of two GQs.
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3.3 GQ folding limits overhang accessibility to complimentary DNA
Figure 8 (A) Schematic of complementary 12-mer, C2, binding to single stranded
portions of the telomeric overhang repeats. (B) Frequency count histograms before
and after binding of C2 to G4, G6, and G3TTAG. (C) Percent accessibility calculated for
single molecule and ensemble FRET. (D) Example contour plots (side view) and (E)
top-down view of G4 molecules and G6 molecules imaged with atomic force
microscopy (AFM). (F) Width distribution histograms of molecules for G4 and G6
before and after the addition of C2 collected by AFM imaging.
A short antisense strand of the telomeric sequence is frequently used to test
unfolding of the GQ(71, 73). In addition, antisense telomeric DNA exists in ALT cells
in the form of c-circles and has been employed as a specific and quantifiable marker
of ALT activity in vivo (74) We applied a similar 12 nt complementary antisense
(CCCTAA)2, C2, in excess to test the accessibility of the G4-G8 overhangs. Binding of
C2 is expected to result in FRET decrease (Fig. 8a). FRET histograms were prepared
from data collected before and after C2 incubation, shown in dark gray and light
blue, respectively (Fig. 8b). As a positive control, we included G3TTAG, a G4 devoid
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of the two terminal Gs, that is unable to fold into GQ (75). The histogram for G4
remained nearly unchanged while G6 and G3TTAG showed a substantial shift after
C2 incubation (Fig. 8b). Due to the multiple conformations of G6 itself, there are
multiple FRET states resulting from C2 binding. The percent accessibility was
calculated by subtracting the histogram before C2 from the histogram after the
addition of C2, indicating the percentage of molecules that had C2 bound (Fig. 9).
Figure 9 FRET histograms are plotted for DNA-only (FRETDNA) and after addition of C2 (FRETDNA+C2). The
∆(delta FRET) is calculated by subtracting the FRETDNA histogram from the FRETDNA+C2 histogram. The
percent accessibility is calculated by summing the portion with the negative FRET change (dark fuschia)
and dividing by the sum of the relative frequencies of the starting histograms within the blue shaded
area.
19
The same experiment was performed at an ensemble level using a fluorometer, from
which the accessibility was calculated by the % change in FRET value before and
after C2 addition (Fig. 10a). The same pattern of accessibility emerged from the two
sets of data, both pointing to a limited accessibility in G4 and G8 and increased
accessibility for G5-G7 in the order of G7>G6>G5 (Fig. 8c, Fig. 10b).
Figure 10 (A) Example of ensemble FRET data taken by fluorometer for G3TTAG with the addition of C2.
The black arrow indicates when C2 is added to G3TTAG FRET DNA. % accessibility for ensemble FRET
measurements is calculated by the formula shown in the figure. The data for all DNAs are included as
part of Figure 2c (light blue bars). (B) Additional single molecule histograms before and after the
addition of C2 to G5, G7, and G8. They are included in Figure 2c (dark blue bars).
As evident from the data, G3TTAG represents the most accessible substrate due to
the lack of GQ folding. Furthermore, we examined atomic force microscopy (AFM)
images of G4 and G6 pre-incubated with C2 (Fig. 8d). In both images, each peak
represents GQ folding. We confirmed that C2 binding only occurs in G6, visualized as
an extended arm from the center peak which arises from the double stranded DNA
formed by C2 binding (Fig. 8e). The peak width of molecules collected from G4 and
20
G6 images also indicates the presence of molecules with increased widths in G6 but
not in G4 (Fig. 8f). For consistency, we used 50-100 mM KCl buffer conditions in all
experiments presented in this study, and all FRET histograms were prepared from
data collected 10 minutes after the proteins were added to DNA substrates (see
Method).
3.4 RAD51 and RPA binding depends on repeat number
Figure 11 (A) Schematic of RAD51 and RPA addition to DNA. (B) Frequency count histogram before and
after addition of RAD51 on G4, G6, and G3TTAG. (C) Molecule frequency count histogram before and
after the addition of RPA to G4, G6, and G3TTAG. (D) Gel image from electrophoretic mobility shift assay
(EMSA) showing binding of RPA to increasing telomeric repeats. The DNA + RPA band is indicated with
the purple arrow, while unbound DNA is indicated with the black arrow. (E) Plot of percent accessibility
calculated from single molecule FRET histograms and percent-bound obtained from EMSA. (F) Summary
plot comparing percent accessibility between RAD51, RPA, and C2 for increasing repeat numbers of the
telomere overhang
21
RAD51 is a key player in the homology-directed repair that triggers ALT (76) .
Furthermore, the inhibition of RAD51 synthesis leads to telomere erosion and
chromosome fusion, suggesting an important role of RAD51 in telomere
maintenance (77) . We sought to examine RAD51’s ability to bind and resolve the
GQ containing telomeric overhang (Fig. 11a). RAD51 addition to G6 and G3TTAG
induces lower FRET molecules as expected from RAD51 binding and filament
formation, whereas the high FRET G4 molecules mostly remain unchanged (Fig.
11b). The percent accessibility resulting from all overhang lengths (Fig. 12a) shows
an overall resemblance to C2 with the largest difference observed in G8 (Fig. 11f,
orange bars). Unlike the case of C2 binding, G8 yields high accessibility to RAD51.
Furthermore, G4 and G5 also exhibit higher accessibility to RAD51 than to C2, likely
due to the small site size of RAD51. RAD51 is a structural homolog of the E. coli
RecA recombinase that engages 3 nt (78) of DNA per protein monomer at a time to
form a filament (79) (80) (81). Therefore, the higher accessibility shown for RAD51
than C2 can be explained at least in part by the different length of overhang required
for RAD51 (3 nt) compared to C2 (12 nt) binding.
22
Figure 12 Additional single molecule histograms before and after the addition of RAD51 (A) and RPA (B)
to G5, G7, and G8 All data are included as part of Figure 3f.
Replication protein A (RPA) is a single strand DNA binding protein that plays a
central role in replication, recombination, and repair. It is thought to contribute to
the assembly of the recombination machinery, which may facilitate the ALT
pathway (82), RPA can unfold telomeric GQ to allow binding of other telomeric
components (83) (84) and plays an important role in telomere maintenance. Based
on its potential function in telomere processing, we investigated whether RPA
binding is modulated by the length of the telomeric overhang (Fig. 11a). An earlier
study revealed that the RPA binding to GQ DNA was limited (29). In agreement, our
accessibility assay revealed limited binding of RPA to G4 and G8 whereas G3TTAG,
G5-G7 (Fig. 12b) showed substantially higher binding (Figs. 11c, f, purple bars). This
is consistent with the footprint size of RPA, i.e. the initial binding mode that involves
8-10 nt and later switches to stable binding to 30 nt (85). Based on the stable
binding expected from RPA, we further quantified the accessibility by performing
electrophoretic mobility shift assay (EMSA) with the same set of DNA substrates.
23
The gel image was analyzed by Image J from which we obtained the fraction of
protein-DNA complex over the sum of the DNA and protein-DNA. The result from
EMSA matches the percent accessibility from the FRET histogram analysis (Fig. 11d,
e). From this result, we conclude that RPA binding, similar to C2, is modulated by
the accessibility of telomeric overhang which is governed by the repeat number (Fig.
11f). In this regard, loading of both RPA and C2 depends on the overhang repeat
number and conformational dynamics.
3.5 WRN and BLM display repetitive unfolding of GQ
Figure 13 (A) Schematic of addition of WRN to DNA. (B) Molecule frequency count
histogram before and after addition of WRN for G4, G6, and G3TTAG. (C) Single
molecule FRET trace of WRN on G4, G6, G3TTAG (D) Summary of percent accessibility
for C2 and proteins including WRN and BLM on increasing telomere repeat numbers.
Human RecQ-family helicases Werner (WRN) and Bloom (BLM) can unwind GQs in
vitro, with high specificity (86) (87). BLM, in conjunction with RAD51 can interact
24
with TRF2 in ALT-activated cells to promote telomere elongation (88), while WRN is
directly involved with telomeric recombination in the ALT pathway (89). WRN and
BLM require a 3' ssDNA tail to unwind a G-quadruplex (89). We subjected both
proteins to varying lengths of telomeric overhangs such that unfolding of GQ could
be detected as FRET decrease (Fig. 13a, Fig. 14a). For the histograms collected
before and after WRN addition, we observed that G4 remained the same, yet G6 and
G3TTAG showed a clear shift (Fig. 13b, Fig. 14b). The same pattern was also seen for
BLM (Fig. 14c).
Figure 14 (A) Schematic of WRN and BLM addition to the telomeric overhang DNA. (B) Additional single
molecule histograms before and after the addition of WRN and ATP to G5, G7 and G8. The data is
presented as part of Figure 4d (green bars). (C) Frequency count histograms before and after addition of
BLM and ATP to G3TTAG and G4 to G8 DNA. The data are include as part of Figure 4d (purple bars). (D)
Like WRN, single molecule traces of the telomeric overhang in the presence of BLM and ATP show fast
FRET fluctuations on G3TTAG and G6, but not on G4 due to limited accessibility.
25
Representative single molecule traces of WRN for G6 and G3TTAG showed a
characteristic FRET fluctuation that lasted over thirty seconds whereas G4 exhibited
a steady high FRET with occasional short-lived drops (Fig. 13c). The same FRET
fluctuation was also seen for BLM (Fig. 14d) and this fluctuation only occurred in
the presence of ATP for both proteins. We interpret this pattern as arising from a
repetitive unfolding of GQ by WRN and BLM. Previously, our work reported on
various helicases that translocate and unwind repetitively on DNA or RNA
substrates (90, 91) (32) (33), and such mode of action was shown to reduce and
prevent other proteins from binding and accessing the single stranded nucleic acid.
Likewise, the repetitive unfolding activity displayed by WRN and BLM may
contribute to keeping the single strand overhang clear from DNA repair proteins, for
example. The percent accessibility for WRN and BLM calculated from FRET
histogram analysis for all DNAs was plotted in addition to C2, RAD51 and RPA (Fig.
13d). With the exception of RAD51 binding to G8, the binding of all molecules tested
exhibit a pattern of limited accessibility to G4 and G8 and enhanced binding to G5 to
G7 and G3TTAG control substrate. We note that the accessibility calculated for WRN
and BLM may be underestimated due to the fluctuating FRET traces that contribute
to broadening of FRET distribution, which increases the overlap between the two
histograms.
26
3.6 POT1 binding is independent of repeat number
Figure 15 (A) Schematic of POT1 unfolding the telomeric DNA. (B) Molecule frequency
count histograms before and after addition of POT1 for G3TTAG and G4 to G8 DNA.
(C) Plot of percent accessibility for POT1 binding to increasing number of telomere
overhang.
Protection of Telomeres-1 (POT1) is a member of shelterin complex that guards and
controls the single stranded telomeric overhang (38). POT1 engages with the
telomeric overhang in a sequence specific manner (TTAGGGTTAG) as clearly
demonstrated by the high resolution structure and biochemical characterization
(92). Recently, we reported that POT1 binds and thereby unfolds GQ in a stepwise
fashion in which one monomer binds TTAGGGTTAG in two successive steps(75).
27
Since the 150 mM NaCl condition used previously may provide a more accessible
binding condition to POT1, here, we applied 50 mM KCl, to promote a more stable
GQ folding. We applied POT1 (200 nM) to the varying length of telomeric repeats
such that successful POT1 binding should result in a FRET decrease (Fig. 15a).
Unlike all the other proteins used above, POT1 binding occurred to a similar degree
with all overhang lengths (Figs. 15b, c). In contrast to the repeat number dependent
loading exhibited by C2, RAD51, RPA, WRN and BLM, POT1 displayed a binding
mode that was independent of the inherent conformational states governed by
repeat number.
3.7 Telomerase binding and extension activity depends on repeat number
Figure 16 (A) Schematic of single molecule pull-down of overexpressed telomerase
(flag-tagged) from HEK293T cells. (B) Images and (C) quantification of molecules
pulled down demonstrating the specificity of telomerase capture. (D) Schematic of
colocalization of telomerase (Alex 647 labeled antibody) and Cy3-labeled telomeric
28
DNA. (E) Percent occupancy of DNA to telomerase calculated by the percent of
colocalization. (F) Gel image from primer extension assay with various repeat length
constructs. (G) Quantification of extension activity by using Image J and Image Quant.
GQ structures inhibit telomerase activity (93) (9), hence the anti-cancer therapy
efforts in developing drugs that stabilize GQ. The sequence specific binding of
telomerase to the telomere is achieved by the complementarity between the
telomeric overhang and the RNA template embedded in the catalytic site of
telomerase (94). In this respect, telomerase shares similarity with POT1. To
examine telomerase activity we used an established telomerase isolation protocol.
HEK 293T cells were transfected with flag-tagged telomerase overexpression
plasmids (generous gift from Tom Cech) (95). The cell lysate was applied to a single
molecule surface pretreated with anti-flag antibody (α-flag) to pull down the flagtagged telomerase(37). The pull-down efficiency was tested by applying the primary
antibody against telomerase followed by red (Alexa 647) labeled secondary
antibody (Fig. 16a). The red fluorescent spots appeared only when all the
components were present and were not detected when any of α-flag, cell lysate or
primary antibody was omitted, indicating the highly specific capture of telomerase
(Figs. 16b, c). We used this platform to evaluate overhang accessibility by applying
green (Cy3) labeled DNA followed by the primary and red labeled secondary
antibody used above (Fig. 16d). By co-localizing the green and red signals, we were
able to count the number of telomerase molecules occupied by the corresponding
DNA. The fraction of red molecules that co-localize with green was used to calculate
the percent occupancy (Fig. 16e). The resulting histogram reveals the length
dependent pattern that was seen for the previous proteins excluding POT1. The
29
limited binding of G4 and G8 to telomerase combined with the improved binding of
G5 to G7 points to a loading mechanism of telomerase that relies on the accessibility
of the telomeric overhang.
Next, we asked if the telomerase extension activity was correlated with the length
dependency reflected in the occupancy shown above. We performed a modified
version of primer extension assay in which flag-telomerase was
immunoprecipitated onto α-flag coated beads and the telomere extension was
stimulated by adding the individual DNA template and mix of dNTPs including
radiolabeled dGTP. The reaction was carried out in the same buffer condition used
for the single molecule assay at 37°C for one hour until the reaction was quenched
and analyzed by 12% PAGE (Fig. 16f). We quantified the product of telomerase
extension by Image J and ImageQuant software. The total intensity collected for each
repeat number variant was normalized by the total intensity obtained for G3TTAG, a
positive control DNA template that leads to the highest extension activity (96) (95)).
As shown, the telomerase extension also exhibits the similar length dependency
which encompasses limited activity for G4 and G8 coupled with a significantly
higher activity measured for G5-G7 (Fig. 16g).
3.8 Discussion
Early work suggested that the formation of GQ at the telomeric overhang limits
access of telomerase and other telomere binding proteins (97) (98) (99) (100)
(Burger et al., 2005; Gomez et al., 2006a; Gomez et al., 2006b; Phatak et al., 2007;
30
Tahara et al., 2006). Furthermore, DMS footprinting study demonstrated that the GQ
preferentially forms at the end of a 3’ overhang rather than at the internal positions,
suggesting that the GQ formation at the end position may inhibit telomerase and
ALT (Tang et al., 2008). We sought to quantitate the accessibility of telomeric
overhang to proteins that are implicated in telomere processing. The single
molecule FRET employed here is an ideal approach in several respects. First, the
range of distance change induced by GQ folding and unfolding is within the FRET
sensitive detection range. Second, single molecule detection enables the resolution
of various conformers of GQ exhibited by the different overhang lengths. Although
we cannot identify the exact conformations represented by different FRET values,
the overall histogram obtained for G4, G5, G6, G7-G8 reveals one, two, four and
multiple states, respectively, as can be expected from alternate pairings of G-triplets
(Fig. 7b). Third, single molecule traces reveal molecular dynamics of GQ as it
undergoes conformational changes by itself (Fig. 7c) or induced by WRN and BLM
(Fig. 13c, Supplementary Fig. 14d). Individual single molecule traces of G5-8 reveal
that they are in dynamic exchange with alternate conformers and that the rate of
exchange is accelerated as the repeat number increases (Figs. 7c, d). While the
conformers appear to be evenly distributed amongst different conformers in G5-G7,
G8 displays a pronounced high FRET peak which exceeds the other states (Fig. 1b).
We interpret this high FRET as arising from two GQs forming on G8, which in turn
yields low accessibility to several proteins tested herein. It is interesting to note that
G8 exhibits both the high propensity for folding and the fast dynamic behavior.
31
While small proteins such as RAD51 gains access to such construct, most other
proteins had a significantly reduced access.
Taken together, we have provided evidence that GQ folding at the 3’ end of
telomeric overhang plays a critical role in controlling protein access. All ALT
associated proteins tested here show the length-dependent loading that resembles
C2 binding to G4-G8. In contrast, POT1 bound the G4-G8 substrates with equal
efficiency, clearly indicating a different binding mode that leads to active disruption
of GQ. A better understanding of the mechanisms for regulating chromosome end
structure and accessibility will help identify new targets for anti-cancer therapies,
with improved specificity for telomeres. Furthermore, our single molecule platform
provides a screening tool for testing the current and future generation GQ ligands,
thereby assessing anti-cancer drugs with single molecular sensitivity
3.9 Methods & Materials
Table 1: DNA Constructs
3’ Cy3 top strand
G3TTAG
TGG CGA CGG CAG CGA GGC TTA GGG TTA GGG TTA GGG TTA G
/3Cy3/
G4
TGG CGA CGG CAG CGA GGC TTA GGG TTA GGG TTA GGG TTA GGG
/3Cy3/
G5
TGG CGA CGG CAG CGA GGC TTA GGG TTA GGG TTA GGG TTA GGG
TTA GGG /3Cy3/
32
G6
TGG CGA CGG CAG CGA GGC TTA GGG TTA GGG TTA GGG TTA GGG
TTA GGG TTA GGG /3Cy3/
G7
TGG CGA CGG CAG CGA GGC TTA GGG TTA GGG TTA GGG TTA GGG
TTA GGG TTA GGG TTA GGG /3Cy3/
G8
TGG CGA CGG CAG CGA GGC TTA GGG TTA GGG TTA GGG TTA GGG
TTA GGG TTA GGG TTA GGG TTA GGG /3Cy3/
18mer
5Cy5/GCC TCG CTG CCG TCG CCA /3Bio/(annealed to all the Cy3
bottom (for
sequence listed above)
FRET)
18mer
GCC TCG CTG CCG TCG CCA /3Bio/(annealed to all the Cy3 sequence
bottom (for
listed above)
pull down)
C2
CCCTAACCCTAA
Table 1 (cont.)
Oligonucleotides (purchased from IDT DNA) were dissolved in 50mM NaCl, 10mM
Tris pH 7.5. Partial duplexed telomeric DNA constructs were prepared by mixing the
3’ Cy3 sequence with the bottom strand at a ratio of 1:1.5 3’Cy3 : bottom strand,
incubated at 95oC for 2 minutes, and slowly cooled to room temperature for at least
2 hours.
Ensemble C2 Measurements
Bulk measurements were performed at room temperature (24±1°C) in a standard,
G-quadruplex formation reaction condition: 100mM KCl, 100mM Tris, with a 10nM
33
partial duplexed telomeric DNA sample. The reaction was initiated with 1nM of C2
DNA. FRET efficiency, E, was monitored with a fluorescence spectrophotometer
(Cary Eclipse, Varian). Accessibility of G-quadruplex DNA was determined by
comparing DNA-only FRET efficiency, E_DNA, with steady-state efficiency after C2
addition, E_(C2+DNA) according to: (E_DNA-E_(C2+DNA))/E_DNA .
Single Molecule Fluorescence Data Acquisition
Single molecule fluorescence experiments were carried out on quartz slides
(Finkenbeiner). Quartz slides and coverslips were coated with polyethylene glycol
(PEG) polymer to minimize non-specific surface interactions(Roy et al., 2008).
Briefly, the slides and coverslips were treated with methanol, acetone, potassium
hydroxide, burned, treated with aminosilane, and coated with a mixture of 97%
mPEG (m-PEG-5000, Laysan Bio, Inc.) and 3% biotin PEG (biotin-PEG-5000, Laysan
Bio, Inc).
Partial duplexed telomeric DNA molecules were immobilized on the PEG-passivated
surface via biotin-neutravidin interaction. Excess donor molecules were washed
away with reaction buffer and supplemented with an oxygen scavenging system (0.8
mg/ml glucose oxidase, 0.625% glucose, ∼3mM 6-hydroxy-2,5,7,8tetramethylchromane-2-carboxylic (Trolox), and 0.03 mg/ml catalase). Imaging was
initiated before and after the protein of interest and in some cases after 20 minutes
of incubation. Experiments and measurements were carried out either at room
temperature (24°C ± 1°C) or at 37°C ± 1°C.
34
A prism type total internal reflection microscopy was used to acquire single
molecule FRET data. A 532-nm Nd:YAG laser was guided through a prism to
generate an evanescent field of illumination. A water-immersion objective was used
to collect the signal and a 550-nm long pass filter was used to remove the scattered
light. Cy3 and Cy5 signals were collected using a 630-nm dichroic mirror and sent to
a charge-coupled device camera. Data were recorded with a time resolution of 100
ms as a stream of imaging frames and analyzed with scripts written in interactive
data language to give fluorescence intensity time trajectories of individual
molecules.
smFRET Data Analysis
Basic data analysis was carried out by scripts written in Matlab, with FRET
efficiency, E, calculated as the intensity of the acceptor channel divided by the sum
of the donor and acceptor intensities. Histograms were generated using over 5000
molecules collected and the relative frequency of the FRET states was plotted. They
were fitted to Gaussian distributions using Origin 8.0 with the peak position left
unrestrained.
Accessibility percentages were calculated using by first normalizing to relative
frequencies and then subtracting the histogram DNA after addition of C2 or proteins
from the DNA-only histograms. (E_DNA-E_(C2/proteins+DNA) ). Then the bars that
35
showed a negative difference were summed and normalized to the area under the
DNA only populations (Fig. 9).
Dwell times were collected by measuring the time the molecule spends in a
particular FRET state. They were then fitted to an exponential decay and the half
times graphed (Fig. 7d). The means and the standard errors were plotted. Scripts
for single-molecule FRET data analysis can be downloaded from
https://physics.illinois.edu/cplc/software/.
AFM Sample Preparation and Imaging
DNA substrates were diluted to 2ng/uL in reaction buffer with additional 10 mM
MgCl2. All samples were incubated at room temperature for 15 minutes.
Samples
were deposited on freshly cleaved mica (Ted Pella, Redding, California), followed by
a wash with deionized water and drying with nitrogen. Imaging was performed on a
Cypher AFM (Asylum Research, Santa Barbara, CA) in tapping mode. AC160TS AC
mode silicon probes with spring constants of ~42 newtons/m and resonance
frequencies of 300 kHz were used. Images were captured at a scan size of 1 um by 1
um with a resolution of 1024 by 1024 pixels.
Protein Purification and Reaction Conditions
RAD51 was purified in a similar way described previously (101). Single molecule
experiments were done at 37C and used 1uM RAD51 with reaction buffer containing
50mM KCl, 1mM MgCl2, and 1mM ATP. The purified hetrotrimer RPA was a kindly
36
provided by Dr. Walter Chazin (Vanderbilt University) (102) .The reaction used 1uM
RPA with 100mM KCl and 10 mM Tris. WRN proteins were purified as described
previously (103). Reactions were performed in 25mM KCl, 1mM MgCl2, 10mM Tris
pH 7.5, and 2mM ATP. BLM was purified as described previously(104) and single
molecule reactions were performed with 100nM BLM in the presence of 3mM MgCl2
20mM Tris pH 7.6, 50mM KCl, and 2mM ATP.
Gel Mobility Shift assay
10nM partial duplex telomeric overhang was mixed with and without 200nM RPA
with Cy5 dyes at the junction and incubated for 15 minutes at 37C with 2mM MgCl2,
10mM Tris pH 7.6, and 50mM KCl. The reaction mixture was loaded and ran on a 6%
acrylamide gel 65V for 2 hours with 0.5x TBE running buffer. Analysis in Image J
was used to quantitate accessibility percentages by taking the area of DNA with RPA
and normalizing it to the areas of the sum of DNA with RPA and DNA alone.
Telomerase overexpression
Over-expression of telomerase was carried out using modification to an established
protocol (105) . Cell lysate containing recombinant telomerase was reconstituted in
HEK293T cells by over-expressing hTR and 3xFLAG tagged hTERT genes in pBS-U1hTR and pVan107 respectively (generous gifts from Dr. Thomas Cech). Cells were
grown to 90% confluency containing DMEM medium (Gibco) supplemented with
10% FBS (Gibco) and 1% Pen Strep (Gibco) at 37°C and 5% CO2. Cells were
37
transfected with 10µg of pBS-U1-hTR plasmid and 2.5 µg pVan107 plasmid diluted
in 625 µl Opti-MEM (Gibco) using 25 µl Lipofectamine 2000 (Invitrogen) diluted in
625 µl Opti-MEM according to the manufacturer’s instruction. Cells were allowed to
grow for 48 hours post-transfection, at which point they were trypsinized and
washed with phosphate buffered saline and lysed using 500 µl Chaps lysis buffer
(10mM Tris-HCl, 1mM MgCl2, 1mM EDTA, 0.5% CHAPS, 10% Glycerol, 5mM βMercaptoethanol, 120U Promega RNasin plus, 1µg/ml pepstatin, aprotinin,
leupeptin, chymostatin, and 1mM AEBSF). Cell lysate was then either flash frozen in
liquid nitrogen and stored at -80°C, or immunopurified.
Immunopurification of telomerase
Telomerase enzyme was immunopurified from cell lysate using ANTI-FLAG M2
affinity gel (Sigma). Prior to addition of the affinity gel to the cell lysate, 40µl of
affinity gel beads were washed three times with 40µl of 1x human telomerase buffer
(50mM Tris-HCl pH8, 50mM KCl, 1mM MgCl2, 1mM spermidine, 5mM βMercaptoethanol, and 30% glycerol) by centrifugation at 4000 RPM for 1 minute at
RT followed by removal of the 40µl supernatant. Following washes, 80µl of affinity
gel-human telomerase buffer slurry was added to the 500µl cell lysate and placed on
a rotator for 3 hours at 4°C. After centrifugation at 4000 RPM and removal of liquid
the beads were washed three times with 1x human telomerase buffer, and flash
frozen in liquid nitrogen as 80µl of bead slurry in 1x human telomerase buffer and
stored at -80°C.
38
Single Molecule Pull-down of Telomerase and Co-localization with Telomeric
DNA
Telomerase is particularly a difficult protein to purify using conventional methods.
We utilized single molecule pull-down methods to pull down the telomerase
complex from over expressed telomerase cell lysate(36). Telomerase was isolated
on the biotinated-PEG coated quartz surface in the following order: Streptavidin,
1:200 antiflag antibody (Sigma, mouse M2, F9291), 1:200 cell lysate, 1:100 primary
hTERT antibody (Abcam, rabbit monoclonal), and 1:10,000 secondary antibody
labeled with Alexa647 (Jackson Immunoresearch, donkey anti-rabbit). Each step
was incubated for 15-30 minutes and 200uL of telomerase reaction buffer (50mM
Tris-Cl pH8, 50mM KCl,1mM MgCl2) was flowed in to wash out unbound molecules
in between each addition. The channels without anti-flag antibodies, primary
antibodies, or cell lysate showed <50 fluorescent spots per imaging area of 2500
μm2, possibly due to surface impurities.
To check the binding affinity of telomerase to DNA, 1nM of the partial duplex
telomeric DNA labeled with Cy3 was incubated with the isolated telomerase on the
surface prior to the addition of antibodies. It was then washed before the hTERT
primary and Alexa647 secondary antibodies were added.
Telomerase extension assay
39
The telomerase extension assay was a modification of a previously described
protocol (106) . The telomerase extension reaction mixture (20µl) contained 1x
human telomerase buffer (described above), 2µl of 3000 Ci/mMole 32P-dGTP
(Perkin Elmer), 0.058mM dGTP, 10mM dTTP, 10mM dATP, 1uM telomeric partial
duplex DNA substrate and 6µl immunopurified telomerase complex. Reactions were
incubated for 1 hour at 30°C, then stopped with the addition of 2µl 0.5M EDTA and
heat inactivated at 65°C for 20 minutes. 8.0 fmol of a 37-mer loading control was
added prior to purification of the sample using an Illustra microspin G-25 column
(GE Life sciences) followed by the addition of 15µl of loading buffer (94%
formamide, 0.1× TBE, 0.1% bromophenol blue, 0.1% xylene cyanol) to 15µl of
sample. The heat denatured (10minutes, 100°C) samples were loaded onto a 10 %
denaturing acrylamide gel (8M urea, 1xTBE) for electrophoresis. 32P incorporation
was imaged using a phosphor screen and phosphorimager (GE Healthcare). Total
activity was quantitated using ImageJ and ImageQuant and normalized to a loading
control as established previously(36) (95). Briefly, each band is individually
quantitated, summed, and normalized to the activity seen with the G3TTAG
construct.
40
IV.
POT1-TPP1 REGULATES TELOMERIC OVERHANG STRUCTURAL
DYNAMICS
The work in this chapter has been published. It is reprinted from Structure, vol. 20,
Hwang H, Buncher N, Opresko PL, Myong S, POT1-TPP1 Regulates Telomeric
Overhang Structural Dynamics, 1872-1880, 2012 with permission from Elsevier
(license number: 3206221494025). From the previous chapter, we saw how the
DNA can affect the binding of an array of proteins. Here, we observed that telomerebinding proteins POT1 alone and POT1 and TPP1 directly regulates the DNA Gquadruplex folding.
Human telomeres possess a single-stranded DNA (ssDNA) overhang of TTAGGG
repeats, which can self-fold into a G-quadruplex structure. POT1 binds specifically to
the telomeric overhang and partners with TPP1 to regulate telomere lengthening
and capping, although the mechanism remains elusive. Here, we show that POT1
binds stably to folded telomeric G-quadruplex DNA in a sequential manner, one
oligonucleotide/oligosaccharide binding fold at a time. POT1 binds from 30 to 50,
thereby unfolding the G-quadruplex in a stepwise manner. In contrast, the POT1TPP1 complex induces a continuous folding and unfolding of the G-quadruplex. We
demonstrate that POT1-TPP1 slides back and forth on telomeric DNA and also on a
mutant telomeric DNA to which POT1 cannot bind alone. The sliding motion is
specific to POT1-TPP1, as POT1 and ssDNA binding protein gp32 cannot
recapitulate this activity. Our results reveal fundamental molecular steps and
dynamics involved in telomere structure regulation.
41
4.1 Introduction
Telomeres are nucleoprotein DNA structures that cap the ends of linear
chromosomes to prevent degradation and chromo- some end-to-end fusions caused
by the inappropriate activity of DNA nucleases and repair enzymes (107).Telomeres
are essential for genome stability and cell survival, and defects in telomere
maintenance correlate with human disease including cancers (108). Telomeric ends
in most eukaryotes consist of repeat sequences with guanine base runs on the 30
single-stranded DNA (ssDNA). In humans, the telomeric overhang consists of 50–
200 nucleotides of tandem TTAGGG repeats, which serves as the substrate for
telomere elongation by telomerase (109) (Makarov et al., 1997). The chemical
nature of the G-rich repeats allows for the ssDNA over- hang to fold into Gquadruplex structure that consist of three tetrads of four guanines interacting via
Hoogsteen base pairing (110) (111) (112) (113) .
In humans, the telomeric overhang is bound by POT1 and TPP1, which are the
homologs of ciliate proteins TEBPa and TEBPb, respectively (114) (115) (116).
POT1 binds single-stranded TTAGG GTTAG sequence and prevents the
inappropriate activation of Ataxia-telangiectasia-mutated and Rad3-related kinase
at the 30 telomeric overhang to ensure that the chromosome end is not recognized
as DNA damage(38). Partial loss or reduction of the 30 telomere overhang elicits a
DNA damage response at telomeres in G1 phase of cell cycle(62), indicating a role of
POT1 in protecting the telomere end. TPP1 increases the affinity of POT1 for DNA by
10-fold (115) (116) and also recruits telomerase in vivo (117). RNA interference
42
silencing of either POT1 or TPP1 induces telomere lengthening and chromosomal
instability (118) (119) (120) (65), clearly indicating their role in regulating
telomerase access to the over- hang. In addition, TPP1 partners with POT1 to
enhance telomerase processivity in vitro (115) . However, unlike the ciliate counter
parts, little is known regarding TPP1 or POT1 modulation of telomeric DNA
structure and G-quadruplex dynamics.
Using single molecule fluorescence assays, we determined the molecular mechanism
involved in the interaction between the telomere overhang and its binding proteins,
POT1 and the POT1-TPP1 complex. We find that a POT1 monomer binds the
telomeric overhang in two successive steps whereby one step likely represents an
individual oligonucleotide/oligosaccharide binding (OB) fold engaging with one
repeat sequence. This results in a sequential unfolding of the G-quadruplex in four
steps by two POT1 monomers. We also demonstrate that the POT1 binding direction
is 30 to 50 with respect to the DNA overhang substrate. Surprisingly, the POT1TPP1 complex exhibits a highly dynamic sliding movement back and forth on the
telomeric overhang, which induces continuous unfolding and refolding of the Gquaduplex. The sliding activity we demonstrate here may provide a mechanistic
basis for how POT1-TPP1 serves to enhance telomerase processivity(115).
43
4.2 POT1 binds the telomeric overhang sequentially one OB fold at a time
Figure 17 (A) Schematic of FRET G4 DNA construct. (B) FRET histograms of G4 molecules from reactions
with increasing NaCl concentration show one folded state (marked F) at 75mM NaCl or above. (C) FRET
histograms of G2, G3, and G4 DNA before POT1 addition. (D) FRET histograms of G2, G3 and G4 after
POT1 addition. (E) Representative single molecule FRET trace of G4 after POT1 addition, indicating
stable low FRET state due to stable binding of POT1 to telomere overhang. (F) SDS-PAGE of purified
POT1 and TPP1N showing purity and the expected molecular weight. (G) Three FRET-DNA substrates
were prepared to test if the two FRET steps represent individual OB fold binding. First one has TTAGGG
TTAG, cognate OB1 and OB2 binding sites whereas the second and third substrates possess mutations on
OB1 and OB2 binding site as marked in red. (H) FRET histograms resulting from POT1 binding to three
DNA substrates. (I) Representative single molecule FRET traces from three experiments show clear two
step binding for the first substrate arising from stable OB1 and OB2 binding in succession. FRET
fluctuation seen in the second substrate indicates unstable binding of OB2 to TTAG. One step FRET
decrease in the third DNA reflects a stable binding of OB1 domain to TTAGGG.
The structure of POT1 bound to telomeric DNA reveals extensive contact between
the OB folds of POT1 and the 10 nucleotide binding sequence, TTAGGGTTAG. OB1
tightly engages with the TTAGGG and OB2 binds the 30-terminal TTAG nucleotides,
which induces a sharp 90 kink in the DNA backbone at the interface between the
44
two OB domains (92). We asked if POT1 can bind the telomeric overhang prefolded
in a G-quadruplex. We prepared a 30 overhang substrate of the sequence
(TTAGGG)4, termed ‘‘G4’’ (Table 1), labeled with two fluorescent dyes, Cy3 and Cy5,
at both extremities of the ssDNA for measuring fluorescence resonance energy
transfer (FRET). As a molecular ruler, FRET reports on the folding status of the DNA
since the donor Cy3 dye will be closer to the acceptor Cy5 dye in compact
conformations to yield a higher FRET compared to unfolded forms. We added POT1
(100 nM) to the G4 FRET construct (Figure 17A) in 150 mM NaCl; a condition in
which G4 exists in one folded conformation (Figures 17A and 17B). G4 contains two
POT1 binding sites as marked in gray shadow (Figure 18B, top). POT1 binding to G4
resulted in a stepwise FRET decrease (Figure 18C, top). We observed four steps of
FRET decrease in the majority of single molecule traces, which is also presented as a
transition density plot (TDP) built from over 200 data points (Figure 18D, top). The
TDP is formed by plotting FRET before transition and FRET after transition on yand x axis, respectively. One cluster represents one FRET transition corresponding
to one binding or dissociation event whereby binding and dissociation events are
plotted in the upper and lower half triangles, respectively (Figure 18D). The POT1
binding to G4 shows four steps of monotonic FRET decrease, indicating successive
four-step binding without dissociation. The four steps observed for two monomer
binding sites suggest that one POT1 monomer binds in two steps, likely due to the
two OB folds binding sequentially. The binding is stable over time as illustrated by
the low FRET peak shown in FRET histogram taken at 10–20 min after the protein
addition (Figures S1C–S1E).
45
Figure 18 (A) Schematic of the G4 DNA construct. The strand with four TTAGGG repeats was attached to a
single molecule surface by annealing a complementary biotinylated strand to form a duplex. Gquadruplex folding induces high FRET between Cy3 (green) and Cy5 (red) at both ends of the ssDNA. (B)
Telomeric repeats for G4, G3, and G2 with POT1 binding sites marked in gray. (C) Single molecule traces
of POT1 binding to G4, G3, and G2 show four, three, and two steps of FRET decrease (arrow),
respectively. (D) Transition density plots are built from the FRET value before transition in the y axis
and the FRET value after transition in the x axis. (E) Dwell times taken from the first (dt1) and second
step (dt2) of FRET decrease obtained from the G4 construct with variable POT1 concentrations. See also
Figure S1. Error bars indicate the SEM.
To further test if the stepwise binding of POT1 occurs one OB fold at a time, we
shortened the constructs to create G3 and G2 with three and two TTAGGG repeats,
respectively (Figure 18B). G3 provides one and a half POT1 binding site whereas G2
only allows one monomer binding. We note that G3 and G2 are not expected to form
higher order structures, yet FRET is 0.7-0.8 due to the ion induced conformational
flexibility of ssDNA, which depends on the ssDNA length (72). According to the same
study, the length corresponding to G4 (24 nt) should yield a 0.5 FRET. Therefore, the
high FRET (0.8) we obtained for G4 above likely arises from the formation of Gquadruplex, whereas the high FRET observed for G3 and G2 results from the short
ssDNA tail length. Under the same POT1 binding conditions as the G4 construct, we
obtained three and two steps of FRET decrease for the G3 and G2, respectively
(Figures 18C and 18D). This is consistent with the interpretation that one POT1
46
monomer binds in two steps. We reasoned that if the stepwise change in FRET
arises from individual OB fold association with the DNA, then the dwell time of the
first step (dt1), which represents the initial POT1 binding, but not the second step
(dt2), which involves the second OB domain binding, should depend on the protein
concentration. To test this, we varied POT1 concentration from 25 to 125 nM and
collected dwell times corresponding to the first (dt1) and the second step (dt2) of
FRET decrease (from 75 molecules) as denoted in Figure 18C. As predicted, the
dwell time shows the expected concentration dependence for the first step, i.e more
rapid binding at higher POT1 concentrations, but not for the second step, which
remains constant regardless of the POT1 concentration (Figure 1E). This result
supports the conclusion that POT1 monomer binds in two successive steps, one OB
fold at a time. To further test the individual OB fold binding, we prepared FRET-DNA
constructs with targeted mutations. OB1 mutant (TTTTTT TTAG) and OB2 mutant
(TTAGGG TTTT) DNA constructs (mutated sequence is underlined) were designed
to perturb the binding of OB1 and OB2, respectively (Figure 17G). OB1 mutant
shows FRET fluctuation indicating an unstable binding of the OB2 domain whereas
the OB2 mutant shows only one step FRET decrease reflecting a stable binding of
the OB1 domain (Figures S17H and S17I). This is consistent with the crystal
structure that shows extensive contact between OB1 and TTAGGG bases whereas
OB2 shows much fewer contacts with the TTAG base(92). This data agrees well with
the dwell time analysis (Figure 18E) and further supports our model that POT1
binds one OB fold at a time.
47
4.3 POT1 binds one OB fold at a time
Figure 19 (A) Schematic of the G4 DNA with various dye positions. (B and C) Schematic diagram
representing the dye positions with respect to the individual OB fold binding sites. CT indicates Cterminal domain of POT1. The positions 24, 18, and 6 nt represent the distances between Cy3 and Cy5.
The faint ovals indicate the OB unit bindings that should not induce FRET decrease. The step number
shown in the single molecule traces (C) expected from individual OB fold binding is written above each
oval. Single molecule traces obtained from the three DNA constructs (C). The dt indicates the time
interval between POT1 addition and the first step of FRET decrease. (D) The average dt from each DNA
construct. The longest dt for the 6 nt DNA construct suggests POT1 binding initiates from the 30 to 50
direction. Error bars indicate the SEM. (E) Schematic of a plausible POT1 binding mode.
Next, we asked if POT1 binding initiates from the 30 end or from the duplex
junction. We prepared two alternate substrates that had the identical DNA
composition to the G4 construct, but the Cy3 dye was relocated 18 or 6 nucleotides
from the 5’ Cy5 dye (Figures 19A and 19B). If POT1 binds from the 30 end, we
expect a time delay prior to the first FRET decrease in the two alternate DNAs. If
POT1 binds from the 5’ end, the FRET decrease should occur at about the same time
in all three constructs. For this comparison, we measured the dwell time between
the time of protein addition and the time of first FRET decrease. Figure 19C shows
the representative traces from all three G4 substrates and the average dwell times
48
analyzed from over 70 molecules. The substantially longer dwell times obtained for
the alternate G4 constructs (Figure 19D) clearly indicate that POT1 selectively
initiates binding from the 30 end, likely due to a better accessibility of the overhang.
Together, our data support a model in which one OB fold of monomeric POT1 binds
G4, partially un- folding the G-quadruplex at the 30 end, which allows the second OB
fold to associate with the adjacent loop/repeat in the DNA. In this way, the four
arms of G-quadruplex are expected to unfold sequentially one-by-one while POT1
binds from 30 to 50 direction, one OB fold at a time (Figure 19E).
To check if the stepwise binding and unfolding is specific to POT1, we used a singlestranded binding protein, gp32 from T4 bacteriophage and the G4 FRET construct.
The gp32 protein exhibits a minimum binding size of seven nucleotides, which is
comparable with POT1’s ten nucleotide sequence requirement. In contrast to POT1,
gp32 binding produced a one-step FRET decrease from 0.8 to 0.3, followed by
dissociation. The protein binding and dissociation was observed as FRET
fluctuations between 0.8 and 0.3 as indicated in the single molecule trace and
transition density plot (Figure 20). Therefore, we conclude that the stepwise
binding is specific to POT1.
49
Figure 20 (A) Schematic of G4 FRET DNA and gp32 protein (B) Single molecule trace shows one step
unfolding of GQ when gp32 binds. (C) Transition density plot of FRET before and after transition built
from FRET values collected from about sixty single molecule traces.
4.4 POT1-TPP1N induces dynamic folding and unfolding of telomeric
overhangs
Figure 21 (A) Schematic of unlabeled TPP1N and POT1 on the G4 FRET construct with Cy3 dyes placed
24, 18, 12, and 6 nt from the duplex junction. (B) Single molecule traces collected from all DNA
substrates shows initial FRET decreases representing POT1-TPP1N binding by POT1 recognition of the
50
telomeric overhang sequence, followed by continuous FRET fluctuations of the 24 and 18 nt DNA and
less pronounced FRET changes for the other DNA constructs. We interpret the FRET fluctuations as
dynamic folding and unfolding of G-quadruplex DNA induced by POT1-TPP1N. (C) FRET histograms built
from FRET values collected from 100 single molecule traces that exhibit dynamic folding-unfolding of
unlabeled POT1-TPP1N on the G4 DNA FRET constructs. (D) Dwell time histograms from over several
hundred events of 24 and 18 nt G4 DNA FRET constructs. See also Figure S3. Gaussian fit yields center of
the histogram with the SEM.
To study how POT1-TPP1 complex interacts with the G-quadruplex folded
overhang, we purified a well-characterized truncated form of TPP1, TPP1N, which
retains the OB domains and stimulates POT1 binding to telomeric DNA and
telomerase processivity(115). TPP1N consists of residues 87 to 334; however, the
87 N-terminal amino acids are functionally dispensable in human cells and are not
conserved in orthologs from other organisms (121) (119) (65). In addition, TPP1N
retains interaction with POT1 and telomerase (116). We applied the preformed
POT1-TPP1N (100 nM) complex to the same G4 DNA as used in POT1 binding
(Figure 3A, top). We observed FRET decrease in two steps, which resembled the one
POT1 monomer binding in which the two steps arise from OB1 and OB2 binding
(compare Figure 21B top panel to Figure 18C bottom panel). Unlike the continuous
stepwise FRET decrease seen in POT1 binding, we observed stable FRET for about
1–2 min, followed by fluctuation at mid- to low-FRET range, implying dynamic and
continuous conformational changes within the telomeric overhang induced by the
POT1- TPP1N complex (Figure 21B, top). To further characterize this behavior, we
used three alternate FRET constructs, which had Cy3 to Cy5 distances of 18, 12, and
6 nt (Figure 21A). When the POT1-TPP1N complex was added to the 18 nt DNA, we
observed one step of FRET decrease from high- to mid-level. The one-step FRET
decrease is expected since the dye position is not sensitive to the first OB binding
but only to the second OB binding. This was followed by FRET fluctuations,
51
indicating dynamic folding and unfolding of G-quadruplex DNA, similar to the 24 nt
construct, except at a higher FRET range due to the closer distance between the two
dyes (Figure 21B, second panel). Such fluctuation is less prominent in the 12 and 6
nt DNA, likely due to a reduced degree of unfolding experienced in this region of
DNA (Figure 21B, third and fourth panels). FRET histograms built from over 100
molecules show the overall FRET pattern in the three substrates tested (Figure
21C). To test if the FRET fluctuations arise from the same source, we measured
dwell times (dt) from several hundred events obtained from the 24 and 18 nt
substrates and plotted them as a histogram (Figure 21D). The similar dwell time
distribution in both DNA constructs suggests that the dynamic fluctuation is not
random, but arises from the same activity induced by POT1-TPP1N. The FRET
pattern observed in the four constructs together indicates that the POT1-TPP1N
complex generates dynamic folding and unfolding of the telomeric overhang DNA.
52
4.5 POT1-TPP1N displays dynamic movement on telomeric overhangs
Figure 22 POT1-TPP1N Slides on the Telomeric Overhang near the 3’End (A) Schematic of Alexa 647labeled TPP1N and POT1 on the G4 DNA construct, with the Cy3 dye located 24, 18, 12, 6, and 0 nt from
the duplex junction. (B) Single molecule traces show the initial FRET increase, followed by dynamic
FRET fluctuations reflecting movement of POT1-TPP1N on the G4 DNA. (C) FRET histograms taken from
over 100 single molecule traces that display FRET fluctuation. (D) Dwell times from several hundred
events from the 24 and 18 nt G4 Cy3-DNA constructs. Gaussian fit yields center of the histogram with the
SEM. (E) Schematic of Alexa 647-labeled TPP1N and POT1 on the G2-mut2 DNA construct. (F) Single
molecule trace of POT1-TPP1N (Alexa 647) on G2-mut2 exhibits dynamic FRET fluctuations similar to
the G4 DNA. (G) FRET histogram built from over 100 single molecule traces that exhibit dynamic FRET
fluctuation on the G2mut2 DNA. (H) Dwell times from over several hundred events of sliding on the
G2mut2 DNA construct. See also Figures S4 and S5. Gaussian fit yields center of the histogram with the
SEM.
How does the POT1-TPP1N complex induce the conformational dynamics in the
telomeric overhang? To address this question, we applied POT1 and fluorescently
53
(Alexa 647) labeled TPP1N to the 3’ Cy3 labeled G4 construct to directly visualize
the protein complex on the DNA substrate (Figure 22A, top). The labeling efficiency
was approximately 65% as measured by absorbance spectrophotometer, and the
presence of a single fluorophore on one protein was confirmed by the intensity level
expected from a single dye and one-step photobleaching of Alexa 647 dye. This
enables us to observe an individual POT1-TPP1N complex on a single DNA molecule.
Here, we detected the binding of a POT1-TPP1N complex as an abrupt appearance of
FRET, followed by continuous FRET fluctuation (Figure 22B, top). We note that the
FRET fluctuation is not due to successive binding and unbinding events because the
lowest FRET value is at 0.3–0.4, which is far above the value expected from dissociation of the protein (0.18). Therefore, we reason that the FRET fluctuations arise
from a continuous association between the labeled protein and the 30 terminus of
the G4 DNA. This activity is specific to POT1-TPP1N since Alexa 647 labeled TPP1N
added to G4 only shows an abrupt FRET increase and decrease reflecting a short
binding period of TPP1N to G4. We also confirmed that fluorescence labeling does
not perturb the protein activity by performing an alternative fluorescence assay,
PIFE (Figure 23) (122);
54
Figure 23 (a) Schematic of Alexa647-TPP1N on Cy3-G4 DNA (b) Single molecule traces show transient
binding and dissociation of TPP1N. (c) Unlabeled TPP1N binding to Cy3-G4 DNA (d) Single molecule
trace shows increased intensity when protein binds. This is an alternative single molecule fluorescence
detection termed PIFE (protein induced fluorescence enhancement). (e) Dwell time analysis of both
FRET and PIFE yield similar binding time, which indicates that fluorescence labeling did not perturb the
protein binding to G4-DNA.
Unlike TPP1N, which only transiently associates with G4 (about 2.5 s averaged over
100 events), the POT1-TPP1N complex stays on the overhang for a substantially
longer period (45 s averaged over 100 events). This is likely due to the behavior of
the POT1-TPP1 complex, not individual protein, since POT1-TPP1 forms a stable
complex when bound to telomere overhang DNA (Liu et al., 2004; Wang et al., 2007;
Xin et al., 2007).
To further characterize this sliding effect, we subjected POT1- TPP1N (Alexa 647) to
four alternate substrates, which have Cy3 dye located 18, 12, 6, and 0 nt away from
the duplex junction (Figure 24A). Both the 24 and 18 nt DNA yield a robust FRET
fluctuation exhibiting high- to low-FRET values, whereas the amplitude of FRET
change is substantially reduced in the 12 and 6 nt DNA, with no detectable FRET
55
change in the 0 nt substrate (Figures 22B and 22C). In light of the folding unfolding
dynamics shown on the same DNA overhang (Figure 21B), the FRET fluctuation
seen in the 24 and 18 nt constructs here is likely arising from POT1-TPP1N sliding
back and forth on the G4 DNA. The more pronounced FRET changes seen in the 24
and 18 nt DNA compared to the other three substrates can be attributed to two
sources. First, TPP1N (Alex 647) is expected to be positioned near the 30 end of
telomeric overhang since it interacts with the C-terminal region of POT1 near the
POT1 OB2 fold (119) (65), giving rise to the high FRET. Second, the activity of POT1TPP1 may be more localized to the 3’ end, consistent with the more pronounced
effect of folding and unfolding dynamics seen near the 30 end (Figures 22A and
22B). In addition, the protein dwell times measured for the 24 and 18 nt DNA
(Figure 22D) match closely to each other as well as to the folding unfolding kinetics
of the DNA as shown in Figure 21D, strongly suggesting that this protein sliding
motion is responsible for the folding and unfolding of G-quadruplex DNA.
4.6 POT1-TPP1N Slides on Mutant Telomeric Sequence
A previous study showed that POT1-TPP1N enhanced the telomerase processivity
even on substrates that possessed modified telomeric sequence (95). The mutant
telomeric sequence had two telomeric repeats followed by multiple repeats of the
mutated sequence ‘‘TTTGGC’’ (modified nucleotides are underlined). The mutant
sequence allows binding and activity of telomerase harboring a mutant RNA
template of the same sequence, but POT1-TPP1N cannot stably bind to the 30
mutated repeats on the substrate. We hypothesized that POT1-TPP1’s sliding
56
movement may occur on this substrate and contribute to enhancing telomerase
processivity.
To test this, we adopted a sequence, which had two telomeric repeats followed by
two repeats of the mutated sequence ‘‘TTTGGC,’’ which we refer to as G2-mut2
(Figure 22E). First, we tested POT1 binding to the G2-mut2 construct. POT1
addition resulted in two steps of FRET decrease followed by a plateau at mid-FRET,
indicating that only one POT1 monomer bound to the cognate sequence of two
telomeric repeats, and the mutated sequence remained unbound by POT1 (Figures
24A and 24B). This is consistent with biochemical data that shows he mutant
sequence does not allow POT1-TPP1 binding (95).
Next, we added the POT1-TPP1N (Alexa 647) complex to the G2-mut2 DNA. Here,
we observed FRET fluctuations analogous to the G4 construct, i.e FRET fluctuation
exhibiting high- to low- levels (Figures 22F and 22G). The presence of high FRET in
the single molecule trace indicates that POT1-TPP1N interacts with the mutant
sequence situated at the 3’ region (Figure 22F). The range of FRET fluctuation and
dwell time measured for this substrate (Figures 22G and 22H) are highly analogous
to the pattern observed in the G4 substrate (Figures 22B and 22C, top), suggesting
that the POT1-TPP1N complex slides back and forth on both substrates.
Additionally, when we lengthened the mutant sequence to four repeats (G2-mut4),
we still observed similar FRET fluctuation, albeit at a slightly reduced FRET value,
strongly suggesting that POT1-TPP1N slides even on a longer stretch of the mutant
sequence (Figures 24C–24F).
57
Figure 24 (A) Schematic of G2-mut2 FRET DNA constructs. (B) Single molecule traces that show two-step
FRET decrease representing one POT1 monomer binding to the cognate sequence G2, (TTAGGG)2. (C)
Schematic of Cy3-labelled G2mut4 (TTAGGG)2-(TTTGGC)4 DNA and POT1- TPP1 (Alexa647). (D) Single
molecule traces reveal FRET fluctuations similar to dynamics of POT1-TPP1 on G4 and G2-mut2. (E)
FRET histograms built from 50 single molecule traces which exhibit FRET fluctuations (F) Dwell times of
over two hundred FRET fluctuations
As a control measurement, we subjected POT1-TPP1N (Alexa 647) to a polythymidine (25 nt) substrate and observed no FRET fluctuation (Figures S5A and
S5B). In addition, we substituted POT1 with gp32 (T4 bacteriophage gene product
58
32), which is an ssDNA binding protein that does not stimulate telomerase
processivity (95). In this case, we obtained FRET traces, which indicate binding and
unbinding of TPP1N, but no FRET fluctuations resembling POT1-TPP1N sliding
(Figures 25C and 25D).
Figure 25 (A) Schematic of Cy3 labeled partial duplex T25, Alex-647 labeled TPP1N and POT1. (B) Single
molecule traces show only transient interaction of TPP1N with DNA. (C) Schematic of Cy3 labeled G4
DNA and Alexa 647 labeled TPP1N and gp32. (D) Single molecule traces reveal transient interaction of
TPP1N to DNA.
59
Therefore, we demonstrate that the dynamic sliding motion is specific to the POT1TPP1N complex on telomeric overhang DNA and that POT1-TPP1 can slide even on
the mutated telomere sequence to which POT1 cannot stably bind on its own. Taken
together, we propose a sliding clamp model whereby the POT1-TPP1N slides back
and forth on the telomeric overhang and thereby generates unfolding and folding
dynamics near the 3’ region (Figure 26). In contrast to the sequential binding of
POT1, which sequesters the overhang in an un- folded state, POT1-TPP1N presents a
disparate mechanism involving a dynamic sliding movement.
Figure 26 Proposed Mechanism of POT1- TPP1N Sliding TPP1N-POT1 binds to the telomeric overhang
from the 3’ end in a POT1-dependent manner and exhibits sliding clamp activity by diffusing along the
telomeric overhang near the 3’ end.
4.7 Discussion
The investigation of telomeric overhang DNA dynamics is complicated because bulkphase ensemble studies cannot resolve transient protein-DNA interactions and
inter- and intra-molecular dynamics. Single molecule approaches allow for the real
time detection of nucleic acid structural dynamics as the substrate undergoes
interaction with a protein and protein complexes. Unexpectedly, we observed a two-
60
step binding process of one POT1 monomer to a telomeric overhang (Figures 18D
and 18E). Based on the sequence specificity of POT1 binding to telomeric DNA (Lei
et al., 2004), our results suggest that one OB fold binds one telomeric repeat at each
step. The dwell time analysis, i.e the difference between 24 and 18 nt DNA (Figures
19C and 19D), indicates that the OB2 fold initiates unfolding of the G-quadruplex by
binding to the TTAG nucleotides at the 30 terminus, followed by the OB1 fold
engaging with the second TTAGGG repeat, further unfolding the G-quadruplex
(Figure 19E). Unlike other ssDNA binding proteins, such as RecA and Rad51, the
POT1 structure reveals a sharp turn between the two OB fold domains, generating a
sawtooth-like bending on the DNA (92). The sequential two-step binding of POT1
that we report here may explain how POT1 binding to the heavily structured
telomeric DNA is facilitated by the two-step binding that results in a kinked domain
arrangement.
The POT1-TPP1N interaction with telomeric G-quadruplex DNA revealed surprising
dynamics, which is in striking contrast to the POT1 binding alone. Our data are
consistent with the interpretation that the POT1-TPP1N complex slides back and
forth on the overhang, generating G-quadruplex unfolding and refolding dynamics
near the 30 end of overhang, possibly contributing to the reduced protein-DNA
FRET changes observed when the DNA dye was located near the 50 side of the
overhang, compared to its location near the 30 end (Figure 22B). The same sliding
motion occurred on an overhang containing mutant telomeric sequence at the 30
end (Figures 22F and 22G), demonstrating that TPP1N alters the nature of POT1
61
interaction with telomeric DNA so as to impart mobility, even on the mutant
sequence to which POT1 cannot bind by itself.
Based on our data, we postulate a mechanism in which the POT1-TPP1N complex
engages with the telomeric sequence in a POT1 dependent manner and converts to a
sliding clamp, which diffuses on the telomeric overhang (Figure 26). TPP1N lacks
the interaction domain with TIN2, which tethers POT1-TPP1 to shelterin and the
telomere (123). Thus, POT1- TPP1 (full length) sliding along ssDNA while tethered
to duplex DNA via shelterin may generate an ssDNA loop as proposed previously
(95) .
This sliding activity may partially explain the ability of POT1- TPP1N to enhance
telomerase processivity, even on an over- hang with mutant telomeric sequence. We
hypothesize that the sliding motion of POT1-TPP1N may serve in this capacity in
three ways. First, it may facilitate loading of telomerase at the 30 terminus by
making the 3’ end of the overhang accessible, i.e when the complex slides away from
3’ end, the 3’ tail is exposed to allow for telomerase loading. Second, the mobility of
POT1- TPP1 near the 30 tail may help to retain telomerase on the telomeric
overhang by continuously engaging with the telomerase. Third, the sliding activity
may physically promote the propulsion of the telomerase, thereby enhancing its
translocation along the telomeric overhang. The ability of the POT1- TPP1N complex
to slide along ssDNA may be highly analogous to the proliferating cell nuclear
antigen sliding clamp, which greatly enhances the processivity of DNA polymerases
(124).
62
4.8
Methods and Materials
Buffers
G2, G3, and G4 salt titration buffers consisted of either 0–100 mM KCl or 0–150 mM
NaCl in 25 mM Tris (pH8). POT1 reaction buffer contained 150 mM NaCl in 25 mM
Tris pH 8. For single molecule imaging, 0.8 mg/ml glucose oxidase, 0.625% glucose,
3 mM 6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic (Trolox), and 0.03
mg/ml catalase were added to the buffers.
Table 2: DNA Constructs
3’ Cy3 top strand
G3TTAG
TGG CGA CGG CAG CGA GGC TTA GGG TTA GGG TTA GGG TTA G
/3Cy3/
G4
TGG CGA CGG CAG CGA GGC TTA GGG TTA GGG TTA GGG TTA GGG
/3Cy3/
G5
TGG CGA CGG CAG CGA GGC TTA GGG TTA GGG TTA GGG TTA GGG
TTA GGG /3Cy3/
G6
TGG CGA CGG CAG CGA GGC TTA GGG TTA GGG TTA GGG TTA GGG
TTA GGG TTA GGG /3Cy3/
G7
TGG CGA CGG CAG CGA GGC TTA GGG TTA GGG TTA GGG TTA GGG
TTA GGG TTA GGG TTA GGG /3Cy3/
G8
TGG CGA CGG CAG CGA GGC TTA GGG TTA GGG TTA GGG TTA GGG
TTA GGG TTA GGG TTA GGG TTA GGG /3Cy3/
63
18mer
5Cy5/GCC TCG CTG CCG TCG CCA /3Bio/(annealed to all the Cy3
bottom (for
sequence listed above)
FRET)
18mer
GCC TCG CTG CCG TCG CCA /3Bio/(annealed to all the Cy3 sequence
bottom (for
listed above)
pull down)
C2
CCCTAACCCTAA
Table 2 (cont.)
Oligonucleotides required to make the partial DNA duplex substrates were
purchased from IDT with either Cy3 or Cy5 dyes (Table 2). The G3 construct was
purchased with an amino modifier C6 dT at the 30 end and reacted with NHS-ester
conjugated Cy3 (GE Healthcare). Briefly, 10 mM dye was incubated with 0.15 mM
DNA in 100 mM sodium tetraborate pH 8.5 buffer overnight. The excess dye was
then filtered out using Micro Bio-spin 6 column (Biorad) twice.
Telomeric DNA constructs were prepared by mixing a 30 Cy3 sequence with the 30
biotin sequence at a molar ratio of 1:1.5 in 20 mM Tris-HCl pH 7.5, 50 mM NaCl and
incubating at 95 C for 2 min then slowly cooling to room temperature for 2 hr.
POT1 and TPP1N Protein Purification
Recombinant human POT1 protein was purified using a baculovirus/insect cell
expression system as described previously (Sowd et al., 2008). The hexahistidine
Sumo-tagged TPP1N (amino acids 89–334) construct was kindly provided by Dr.
64
Ming Lei (University of Michigan). Expression was induced with 0.1 mM isopropyl 1thio-b-D-galactopyranoside in Escherichia coli BL21 (DE3) pLysS cells, and the
protein was purified as previously described (125).
Fluorescent Labeling of TPP1N
The Alexa647 (NHS ester) was reacted with TPP1N in a 1:20 protein-dye ratio
for 1 hr in 100 mM sodium bicarbonate buffer, pH 8.5. Excess dye was removed
using a Micro Bio-spin 6 column (BioRad) twice.
Single Molecule Fluorescence Data Acquisition
Single molecule fluorescence experiments were carried out on quartz slides
(Finkenbeiner). To minimize surface interactions with the protein, quartz slides and
coverslips were coated with polyethylene glycol (PEG) (27). Briefly, the slides and
coverslips were cleaned and treated with methanol, acetone, potassium hydroxide,
burned, treated with aminosilane, and coated with a mixture of 97% mPEG (m-PEG5000, Laysan Bio, Inc.) and 3% biotin PEG (biotin-PEG-5000, Laysan Bio, Inc).
Partial duplex DNA molecules were annealed and immobilized on the PEG
passivated surface via biotin-neutravidin interaction. Excess donor molecules were
washed away with reaction buffer and supplemented with an oxygen scavenging
system (0.8 mg/ml glucose oxidase, 0.625% glucose, 3 mM 6- hydroxy-2,5,7,8-
65
tetramethylchromane-2-carboxylic (Trolox), and 0.03 mg/ml catalase). Imaging was
initiated before protein (POT1, TPP1N, or POTTPP1N) was flowed through to
capture the moment of protein binding to DNA. All experiments and measurements
were carried out at room temperature (23C±1C).
Prism type total internal reflection microscopy was used to acquire single molecule
FRET and PIFE data. A 532-nm Nd:YAG laser was guided through a prism to
generate an evanescent field of illumination. A water-immersion objective was used
to collect the signal and a 550-nm long pass filter was used to remove the scattered
light. Cy3 signals were collected using a 630-nm dichroic mirror and sent to a
charge-coupled device camera. Data were recorded with a time resolution of 100 ms
as a stream of imaging frames and analyzed with scripts written in interactive data
language to give fluorescence intensity time trajectories of individual molecules.
smFRET Data Analysis
Basic data analysis was carried out by scripts written in Matlab, with FRET
efficiency, E, calculated as the intensity of the acceptor channel divided by the sum
of the donor and acceptor intensities. Histograms were generated using over 6,000
molecules collected and were fit to Gaussian distributions using Origin 8.0, with the
peak position left unrestrained. Fluorescence resonance energy transfer TDP are
two-dimensional contour maps plotted from transitions collected from 200–800
FRET transitions. TDP is constructed by plotting values for each transition based
66
upon FRET value from which the transition originated (y axis) to which FRET value
the transition ends (x axis). Dwell times were collected by measuring the time the
molecule spends in a particular FRET state. The means and the standard errors
were plotted. Software for analyzing single-molecule FRET data is available for
download from https://physics.illinois.edu/cplc/software.
67
V. A REAL-TIME SINGLE TELOMERASE EXTENSION ASSAY
This chapter contains work that is currently ongoing. Below are some preliminary
but promising results that we have obtained. To date, little information is known
about the processivity of a single telomerase. We have developed an assay that can
measure dissociation constants of telomerase with its substrate as well as how
extension rates of a single telomerase. Addition controls are needed to validate the
assay.
Telomerase is a complex ribonucleoprotein that adds telomeric repeats to the ends
of the chromosome. To directly measure the processivity of a single telomerase, we
have developed an a single telomerase extension assay that utilizes the single
molecule pulldown technique (SiMPull) to isolate human telomerase from cell
lysate. This method employs a Cy3-probe that fluoresces when 3 telomeric repeats
have been extended. We have obtained single molecule traces that provide dynamic
information on assembly time, extension time, and enzyme processivity. We have
also validated our method through counting the number of fluorophores bound at
the end of the experiment, hence measuring enzyme activity. Our experiments
reveal that the translocation and dissociation step regulates the processivity of
telomerase.
5.1 Introduction
Telomerase activity is unregulated and overexpressed in more than 85% of human
tumors, while it is found at relatively low levels in normal human cells. This
68
overexpression and activity makes telomerase an attractive target in cancer
diagnosis and therapy.
Currently, the conventional assay involves incubating telomerase with a telomeric
DNA primer and radiolabelled deoxynucleotide triphosphates in magnesium buffer.
The products are then resolved on a polyacrylamide and the ladder of bands
corresponds to the added products. In practice, there are two types of assays most
frequently used.
The telomeric repeat amplification protocol (TRAP) uses polymerase chain reaction
(PCR) to amplify the telomeric products, which is a highly sensitive technique.
However, the amount of products indicated on the gel readout is dependent of the
amplification step and may not represent the quantity of extension products. Thus,
TRAP is susceptible to amplification related errors, relatively time consuming, and
most importantly, can lead to misinterpretation. The second method is the direct
telomerase assay where biotinylated telomeric substrates are used so that the
reaction products are immobilized on streptavidin-coated beads and washed with
buffer to eliminate excess 32P-dGTP (126) (115). Reactions can then be incubated
for a given amount of time, and the samples denatured with heat and loaded onto a
polyacrylamide with urea and TBE denaturing gel for electrophoresis. With this
method, telomerase activity can be detected directly and is the ideal tool for
analyzing the details of the reaction mechanism.
Alternatively, fluorescent methods can be used to determine telomerase activity.
David Klenerman’s group (University of Cambridge) analyzed human telomerase
activity and function by two color single molecule coincidence fluorescence
69
spectroscopy, employing Cy5-dATP incorporation into a telomerase primer that has
been pre-labelled with a reference fluorophore. When excited with two independent
lasers, the number of extended products generated can be determined (127). Their
assay involves the incorporation of a non-natural deoxynucleotides which may be
rate-limiting. With coincidence fluorescence spectroscopy, it is also difficult to track
one telomerase for an extended amount of time.
Conventionally, processivity of an enzyme is defined as the ability of an enzyme to
act and catalyze consecutive reactions without releasing its substrate (128). In this
case, it would be how many repeats a telomerase can add before it dissociates from
the primer. Current studies have only defined processivity of telomerase in terms of
activity and processivity in terms of length of extension products. Telomerase
processivity measured in vitro is a function of temperature, ionic and dGTP
concentration. The processivity increases proportionally with temperature and is
inversely proportional with salt concentration (129, 130). There are two scenarios
that could occur: one telomerase enzyme could extend a primer continuously or
different telomerase can dissociate, rebind, and extend multiple times. Processivity,
strictly speaking, would be different in between the two, but with a bulk assay or a
gel, it would be impossible to differentiate between the two. These assays are
dependent on concentration of telomerase available in the cell lysate which could
vary due to sample preparation. They are also snapshots of the final product at
different time points and cannot distinguish whether the extension is by one
70
telomerase acting continuously or by multiple events of a single telomerase binding,
extending, and dissociation.
5.2 Development of a Real Time Single Telomerase Extension Assay
We sought out to develop a real-time single telomerase extension assay. Telomerase
can be isolated and immobilized through single-molecule pulldown assay
(SiMPULL) as previously described onto a quartz slide. Telomeric substrate (G3,
sequence (TTAGGG)3) is flowed in and allowed to bind. We start recording and the
dNTP and 10x Cy3-probes are flowed in with imaging buffer. In this manner, we can
study real-time extension as the probes bind to the extended substrate.
Presence of the telomerase in the pulldown can be confirmed separately when
tagged with primary anti-TERT and fluorescent-secondary antibodies. (Refer to
Chapter 3.7).
When we checked the activity of the telomerase through a direct telomerase assay,
we saw that up to 15 repeats (red asterisks) were added through the direct
telomerase assay after incubation for an hour at 30C (Figure 27A). To test the
extension products with the same lysate and substrate (G3), after an hour at 30C, we
photobleached our molecules, and counted steps of fluorescence decrease in the
absence and presence of 500μM dNTP (Figure 27B). In the presence of dNTP, there
was a significant amount of more probes bound (Figure 27C). Without dNTP, we
observe only one probe is able to bind to the substrate. This may be due partial
adequate space on the substrate or nonspecific binding of probe directly to the
surface in the absence of telomerase.
71
As a control, we counted annealed probes and saw a linear relationship and max
binding (not shown). The Cy3 probe has the sequence of CCCTAACCCTAACCC and
can binds between 2 and a half repeats, thus spanning three repeats. Numerically,
we only saw an increase of up to 12 repeats. We explain this being lower due to the
incomplete dye-conjugation or photobleached molecules (85% labeling efficiency).
Figure 27 (A) Direct telomerase assay of gel shows up to 15 repeats of extension. (B) Photobleaching
steps reveal 2 probes of extension. (C) In the presence of dNTP, there are more probes that bind to the
substrate.
5.3 Telomerase assembly is dependent on dNTP concentration
From single molecule traces, we can collect dwell times from the following periods:
wait times, extension times, and bound times (Figure 28A). The wait time is how
long it takes for the telomerase to assemble and extend. The extension time
represents how long it takes the telomerase to extend and the probes to anneal. We
wanted to see how these dwell times would change as we varied dNTP
concentration. When we decreased the dNTP concentration into the uM range, we
observed that only the wait time slows down (Figure 28B). Extension time is similar
across the dNTP in these ranges. This implies the assembly and conformation
change made by telomerase in dnTP-dependent and remains before extension.
72
Figure 28 (A) Dwell times of wait, extension, and bound times can be collected and measured. (B) They
can be fit to an exponential curve and the decay time can be plotted. Error bars representant standard
deviation.
5.4 POT1-TPP1 promotes telomerase extension and dissociation
In 2010, Latrick and Cech proposed that POT1-TPP1 enhances telomerase
processivity by slowing primer dissociation and aiding translocation (95). To test
whether this is true, we applied the real time extension assay in the presence of
POT, and POT1-TPP1. We incubated the substrate with POT1 or POT1-TPP1 and
then stimulated extension by adding dNTP (500uM). We hypothesized that if POT1TPP1 slows primer dissociation, the substrate will stay bound longer. Surprisingly,
we observed from single molecule extension traces (Figure 29A) that the bound
times and extension times are significantly shorter in the presence of POT1-TPP1
(Figure 29B). In addition, when we count the number of probes that can bind in a
single binding event, we observe there are more probes binding and more extension
in the presence of POT1-TPP1 (Figure 29C).
73
Figure 29 (A) Single molecule traces in the absence and in the presence of POT1/TPP1. (B) Wait,
extension, and bound times collected with POT1 and POT-TPP1. (C) Amount of probes that are bound to
the extension products within one binding event.
5.5 Sliding of telomeric overhang POT1-TPP1 in the presence of telomerase
Since POT1-TPP1 seemed to enhance processivity by extending faster and
dissociating faster. We wondered if POT1-TPP1 would induce a similar foldingunfolding on DNA (seen in Chapter 2) in the presence of telomerase. We utilized our
single molecule pull-down assay and used a FRET Cy3-Cy5 labeled G3TTAG
construct. In the absence of POT1-TPP1, single molecule traces show one stable
FRET state and the FRET histograms reflect a high FRET peak (Fig30C). However,
when POT1-TPP1 are added, there is appearance of fluctuations corresponding to
the sliding movement of the POT1-TPP1 on the telomeric DNA (Figure 30D) and the
FRET histogram become broader (Figure 30C,F). As a control, we counted
fluorescence steps and saw very little extension in both conditions (without, with
POT1-TPP1). We attribute the lack of extension to the placement of the Cy3-dye at
the 3’ end of the DNA substrate. Taken together, POT1-TPP1 retains it’s ability to
fold and unfold DNA even in the presence of the telomerase enzyme. We also
74
demonstrate that we can isolate telomerase to the surface and apply FRET
constructs to monitor dynamics.
Figure 30 (A) Schematic of FRET construct on telomerase (B) Single molecule trace of G3TTAG bound to
telomerase in the presence of dNTP. (C) FRET histogram collected from over 50 traces. (D) Schematic of
FRET construct in the presence of POT1-TPP1 with telomerase (E) Single molecule trace showing
fluctuations (F) Summary histogram collected from 50 FRET traces
5.6 Discussion
We have developed an assay that allows the study of the processivity of a single
telomerase. In the presence of excess substrates, we see only a fraction of
substrates, only 40%, which was extended by telomerase (Figure 27C). The overall
activity and processivity of our single telomerase extension assay agrees with the
direct telomerase assay at bulk after an hour. However, this method can provide the
temporal axis on the binding rate as well as processivity of a single enzyme.
We note that since the labeling efficiency of the Cy3-probe is only ~85%, and thus
we are underestimating the number of probes that are observed binding and hence
extended telomeric repeats. In addition, our assay is only a pseudo real-time assay,
75
because it requires at least three repeats on the extended product for the probe to
bind. Our assay reports on the unexpected high rate of dissociation of telomerase
from the extended substrate.
This type of result can explain why previous studies saw a lack of correlation
between disease association and decreased telomerase activity for certain alleles
(131) (132). In those studies, telomerase activity and processivity did not seem to
be affected when mutated TERT was mixed with wildtype TERT. A possible
explanation would be that the binding and unbinding of telomerase rate is so fast,
and that a modest deficit in telomerase activity (20%) would result in a normal
telomere phenotype. Furthermore, our result showing high dissociation rate which
may explain why activity of a variant allele with a wildtype allele in a heterozygote
can be predicted by calculating the average activity (from the wildtype and mutant)
in the context of the heterozygote.
5.7 Methods and Materials
Buffers
All experiments in this chapter were conducted using telomerase extension buffer
50mM Tris-Cl pH8, 50mM KCl,1mM MgCl2. The addition of 0.1 mg/ml bovine serum
albumin (New England Biolabs) was used as wash buffer. Unbound antibodies and
sample were removed from the channel by washing with wash buffer between
additions of antibodies and sample.
76
Table 3: DNA Constructs
G3
TTA GGG TTA GGG TTA GGG
G3TTAG (for TGG CGA CGG CAG CGA GGC TTA GGG TTA GGG TTA GGG TTA GG
FRET)
/3Cy3/
18mer
5Cy5/GCC TCG CTG CCG TCG CCA annealed to all the Cy3 sequence
bottom (for
listed above)
FRET)
Cy3-probe
Cy3-CCCTAACCCTAACCC
Oligonucleotides were purchased from IDT (Table 3). We used single stranded for
the telomerase extension assays, while using the partial duplex substrates for the
FRET experiment. Partial duplex telomeric DNA constructs were prepared by
mixing a Cy3 sequence with the biotin sequence at a molar ratio of 1:1.5 in 20 mM
Tris-HCl pH 7.5, 50 mM NaCl and incubating at 95 C and then slowly cooled to room
temperature.
POT1 and TPP1N Protein Purification
Recombinant human POT1 protein was purified using a baculovirus/insect cell
expression system as described previously (125). The hexahistidine Sumo-tagged
TPP1N (amino acids 89–334) construct was kindly provided by Dr. Ming Lei
(University of Michigan). Expression was induced with 0.1 mM isopropyl 1-thio-b-D-
77
galactopyranoside in Escherichia coli BL21 (DE3) pLysS cells, and the protein was
purified as previously described ((125), see above Chapter 4).
Single Telomerase Fluorescence Extension Assay
Single molecule fluorescence experiments were carried out on quartz slides
(Finkenbeiner). Quartz slides and coverslips were coated with polyethylene glycol
(PEG) (Roy et al., 2008). Briefly, the slides and coverslips were cleaned and treated
with methanol, acetone, potassium hydroxide, burned, treated with aminosilane,
and coated with a mixture of 97% mPEG (m-PEG-5000, Laysan Bio, Inc.) and 3%
biotin PEG (biotin-PEG-5000, Laysan Bio, Inc).
Telomerase was localized to the surface through single molecule pulldown methods
from overexpressed cell lysate (Jain et al, 2011). Biotinated anti-flag-antibodies
(1:200 Sigma, mouse M2, F9291) then 1:50 cell lysate was added. Next, the
substrate 10nM ssG3 DNA was incubated without or with proteins (400nM POT1,
400nM TPP1, or 400nM POT1-TPP1).
Single molecule imaging was initiated before 500uM (or otherwise noted) dNTP
was flowed in with imaging buffer containing an oxygen scavenging system (0.8
mg/ml glucose oxidase, 0.625% glucose, 3 mM 6- hydroxy-2,5,7,8tetramethylchromane-2-carboxylic (Trolox), and 0.03 mg/ml catalase) to capture
the time it takes for telomerase to initiate and elongate and the moment of Cy3probe binding. A prism-type TIRF setup was used with a 532 nm laser to generate
an evanescent field of illumination. Cy3 signals were collected using a 630-nm
dichroic mirror and sent to a charge-coupled device camera. Fluorescence
78
intensities of molecules were recorded with a time resolution of 50-100 ms as a
stream of imaging frame, and analyzed with scripts written in interactive data
language to give fluorescence intensity time trajectories of individual molecules. All
experiments and measurements were carried out at room temperature (23C±1C),
unless otherwise noted.
smFRET Data Analysis
Basic data analysis was carried out by scripts written in Matlab. Histograms were
generated using over 6,000 molecules collected and were fit to Gaussian
distributions using Origin 8.0, with the peak position left unrestrained. Dwell times
were collected by measuring the time before the probe binds after dNTP was added
(wait time), between each additional probe binding (extension), and the time the
probes are bound before dissociating (bound time). Dwell times are fit to an
exponential decay and the half times are plotted with the standard deviation.
79
VI.
CONCLUSIONS AND PERSPECTIVE
The goal of my study is to examine the mechanisms that regulate the telomeric
overhang dynamics and accessibility. This can eventually lead to screening and
identification of new targets for anti-cancer therapies, with increased specificity to
telomeres. We propose that therapies that target the overhang structure, rather
than telomerase itself, may hold greater promise because they can inhibit both
pathways to which cancer cells use to proliferate indefinitely. We showed that the
DNA has regulatory functions on protein binding, while POT1-TPP1 can affect
secondary G-quadruplex structure. Furthermore, we have developed a real-time
assay to test the rate and extent of extension by telomerase.
6.1 Accessibility of telomeric overhang by ALT proteins and telomerase
We observed that most telomere-binding proteins (ALT proteins and telomerase)
can only passively bind and unfold G-quadruplex structures. Unlike the other
proteins, POT1 is able to actively unfold the G-quadruplex. Hence, we can
hypothesize that the G-quadruplex secondary structure inhibits telomerase from the
initial binding step. Furthermore, the G-quadruplex structure may play a physical
role as a last-resort protection by the cell when the telomere length is too short.
6.2 Why does POT1-TPP1 slide on DNA?
From our results, we concluded that POT1 can bind stably and unfold the Gquadruplex, while POT1-TPP1 as a complex induces a sliding motion that may serve
to enhance telomerase processivity. However, some questions still remain
80
unanswered, in particular, how does the sliding mechanism of POT1-TPP1
upregulate telomerase activity and processivity? It is known that the binding affinity
of telomerase to its substrate is increased when POT1-TPP1 is present ((95). Could
the increase in telomerase activity and processivity be due to the accumulation of
more telomerase recruitment by more POT1-TPP1 complexes? Or, could the sliding
of POT1-TPP1 be providing the physical translocation movement of telomerase or is
it to clear off the other proteins from the telomere?
With our newly developed single telomerase extension assay, we can actively
observe the mechanism surrounding the POT1-TPP1 and telomerase system. It is
still unknown if POT1-TPP1 provides additional movement for telomerase to
increase its processivity or if it straightens out the DNA so that telomerase can act.
By monitoring the distance between each of the three proteins, we can begin to
study mechanisms of the Shelterin complex, in particular, POT1-TPP1, during
telomerase extension.
6.3 Telomerase activity can be measured as a function of binding and
extension
Here, we developed a novel method for measuring telomerase processivity, unique
in its ability to measure the processivity of an individual telomerase. This is a
preliminary study, and further experiments are necessary to validate the single
telomerase extension assay. For example, we should titrate the cell lysate
concentration and observe that rate is unchanged, but overall extension by
81
telomerase increases. As a control, we should also test various Cy3-probe lengths to
figure out exact binding size of a probe.
At this time, we do not have a concise explanation to the trend of assembly time is
correlated to dNTP concentration. It is possible that dNTP induces conformational
changes on the telomerase in preparation of extension.
The single telomerase extension assay has potential to become a comprehensive
technique, useful with other substrates to study stalling by G-quadruplees during
telomerase extension and the efficacy of telomerase inhibition by G-quadruplex
stabilizing drugs. While we do not expect this highly sensitive assay to replace the
direct telomerase activity assay, we do feel that it does complement the traditional
technique by providing temporal information of a single telomerase. This would be
useful for studying telomerase from heterogeneous populations and answering
questions about haplo-insufficiency of telomerase mutations.
6.4 Long term goals
The mechanism behind the telomerase extension is still poorly understood. Single
molecule experiments can provide some dynamic information about extension rates
and dissociation constants. To better understand the functional dynamics of the
telomere-protein complex, more single molecule experiments of every component
of the Shelterin and ALT pathway complex should be characterized. With the
development of the telomerase extension assay, one can study the effect of the
substrates and telomere-binding proteins on overhang extension by telomerase.
The long-term goal is that the study of the individual components of the Shelterin by
82
itself and with telomerase can provide insights on the mechanism of telomere
regulation at chromosome ends.
Furthermore, determining how small molecule drugs inhibit telomerase extension
would be valuable in synthesizing a novel drug that can specifically target
telomerase extension. In addition, a single molecule assay may be useful in
developing a platform for screening small molecules for efficacy of inhibition of
telomerase activity
83
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